Sara Gasperini

The neutrophil as a cellular source of chemokines.

Neutrophils are known to play an important role in inflammatory responses by virtue of their ability to perform a series of effector functions that collectively represent a major mechanism of innate immunity against injury and infection. In recent years, however, it has become obvious that the contribution of neutrophils to host defence and natural immunity extends well beyond their traditional role as professional phagocytes. Indeed, neutrophils can be induced to express a number of genes whose products lie at the core of inflammatory and immune responses. These include not only Fc receptors, complement components, cationic antimicrobial and NADPH oxidase proteins, but also a variety of cytokines (including tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-1R alpha, IL-12 and vascular endothelial growth factor), and chemokines such as IL-8, growth-related gene product, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interferon-gamma-inducible protein of 10 kDa and monokine induced by interferon-gamma. Because these chemokines are primarily chemotactic for neutrophils, monocytes, immature dendritic cells and T-lymphocyte subsets, a potential role for neutrophils in orchestrating the sequential recruitment of distinct leukocyte types to the inflamed tissue is likely to occur. The purpose of this review is to summarize the essential features of the production of chemokines by polymorphonuclear neutrophil leukocytes and the contribution that we have made to characterize some aspects of this newly discovered crucial function of neutrophils.

IL-10–induced microRNA-187 negatively regulates TNF-α, IL-6, and IL-12p40 production in TLR4-stimulated monocytes

IL-10 is a potent anti-inflammatory molecule that, in phagocytes, negatively targets cytokine expression at transcriptional and posttranscriptional levels. Posttranscriptional checkpoints also represent the specific target of a recently discovered, evolutionary conserved class of small silencing RNAs known as “microRNAs” (miRNAs), which display the peculiar function of negatively regulating mRNA processing, stability, and translation. In this study, we report that activation of primary human monocytes up-regulates the expression of miR-187 both in vitro and in vivo. Accordingly, we identify miR-187 as an IL-10–dependent miRNA playing a role in IL-10–mediated suppression of TNF-α, IL-6, and the p40 subunit of IL-12 (IL-12p40) produced by primary human monocytes following activation of Toll-like receptor 4 (TLR4). Ectopic expression of miR-187 consistently and selectively reduces TNFα, IL-6, and IL-12p40 produced by LPS-activated monocytes. Conversely, the production of LPS-induced TNF-α, IL-6, and IL-12p40 is increased significantly when miR-187 expression is silenced. Our data demonstrate that miR-187 directly targets TNF-α mRNA stability and translation and indirectly decreases IL-6 and IL-12p40 expression via down-modulation of IκBζ, a master regulator of the transcription of these latter two cytokines. These results uncover an miRNA-mediated pathway controlling cytokine expression and demonstrate a central role of miR-187 in the physiological regulation of IL-10–driven anti-inflammatory responses.

Up-regulation of IL-10R1 expression is required to render human neutrophils fully responsive to IL-10.

We have recently shown that IL-10 fails to trigger Stat3 and Stat1 tyrosine phosphorylation in freshly isolated human neutrophils. In this study, we report that IL-10 can nonetheless induce Stat3 tyrosine phosphorylation and the binding of Stat1 and Stat3 to the IFN-γ response region or the high-affinity synthetic derivative of the c-sis-inducible element in neutrophils that have been cultured for at least 3 h with LPS. Similarly, the ability of IL-10 to up-regulate suppressor of cytokine signaling (SOCS)-3 mRNA was dramatically enhanced in cultured neutrophils and, as a result, translated into the SOCS-3 protein. Since neutrophils’ acquisition of responsiveness to IL-10 required de novo protein synthesis, we assessed whether expression of IL-10R1 or IL-10R2 was modulated in cultured neutrophils. We detected constitutive IL-10R1 mRNA and protein expression in circulating neutrophils, at levels which were much lower than those observed in autologous monocytes or lymphocytes. In contrast, IL-10R2 expression was comparable in both cell types. However, IL-10R1 (but not IL-10R2) mRNA and protein expression was substantially increased in neutrophils stimulated by LPS. The ability of IL-10 to activate Stat3 tyrosine phosphorylation and SOCS-3 synthesis and to regulate IL-1 receptor antagonist and macrophage-inflammatory protein 1β release in LPS-treated neutrophils correlated with this increased IL-10R1 expression, and was abolished by neutralizing anti-IL-10R1 and anti-IL-10R2 Abs. Our results demonstrate that the capacity of neutrophils to respond to IL-10, as assessed by Stat3 tyrosine phosphorylation, SOCS-3 expression, and modulation of cytokine production, is very dependent on the level of expression of IL-10R1.

The MYD88-Independent Pathway Is Not Mobilized in Human Neutrophils Stimulated via TLR4

LPS activates both MyD88-dependent and -independent signaling via TLR4, but the extent to which each cascade is operative in different cell types remains unclear. This prompted us to revisit the intriguing issue of CXCL10 production, which we previously showed to be inducible in neutrophils stimulated with LPS and IFN-γ but not with either stimulus alone, contrary to other myeloid cells. We now report that in neutrophils the MyD88-independent pathway is not activated by LPS. Indeed, microarray and real-time PCR experiments showed that neither IFNβ nor IFNβ-dependent genes (including CXCL10) are inducible in LPS-treated neutrophils, in contrast to monocytes. Further investigation into the inability of LPS to promote IFNβ expression in neutrophils revealed that the transcription factors regulating the IFNβ enhanceosome, such as IFN-regulatory factor-3 and AP-1, are not activated in LPS-treated neutrophils as revealed by lack of dimerization, nuclear translocation, confocal microscopy, and inducible binding to DNA. Moreover, we show that the upstream TANK-binding kinase-1 is not activated by LPS in neutrophils. A lack of IFNβ/CXCL10 mRNA expression and IFN-regulatory factor 3 activation was also observed in myeloid leukemia HL60 cells differentiated to granulocytes and then stimulated with LPS, indicating that the inability of neutrophils to activate the MyD88-independent pathway represents a feature of their terminal maturation. These results identify a disconnected activation of the two signaling pathways downstream of TLR4 in key cellular components of the inflammatory and immune responses and help us to better understand the primordial role of neutrophils in host defense against nonviral infections.

Molecular mechanisms underlying the synergistic induction of CXCL10 by LPS and IFN‐γ in human neutrophils

The CXCL10 chemokine is a critical chemoattractant for the recruitment of activated Th1 and NK cells into inflammatory sites. CXCL10 is typically produced by myeloid cells in response to IFN‐γ, as well as by neutrophils, though the latter require a costimulation with IFN‐γ and LPS. In this study, we investigated the molecular mechanism(s) whereby IFN‐γ and TLR4 ligation synergize to induce CXCL10 expression in neutrophils. By primary transcript real‐time PCR analysis, we demonstrate that the CXCL10 gene is transcriptionally induced by the LPS plus IFN‐γ combination in neutrophils, consistent with previous studies showing that increased CXCL10 gene expression does not reflect enhanced mRNA stability. The IFN‐γ‐induced STAT1 activation and the lipopolysaccharide (LPS)‐induced NF‐κB activation were not enhanced if neutrophils were exposed to both stimuli, whereas both transcription factors were activated by IFN‐γ or LPS in monocytes. Finally, pharmacological inhibitors of NF‐κB demonstrated its role in the induction of CXCL10 expression by LPS plus IFN‐γ in neutrophils, and by LPS or IFN‐γ in monocytes. Together, these results suggest that in neutrophils, the synergy observed between LPS and IFN‐γ toward CXCL10 gene expression likely reflects the cooperative induction of the NF‐κB and STAT1 transcription factors by LPS and IFN‐γ, respectively.

Uncovering an IL-10-dependent NF-κB recruitment to the IL-1ra promoter that is impaired in STAT3 functionally defective patients

The interleukin 1 receptor antagonist (IL‐lra) is an important negative regulator of the inflammatory response, whose genetic deficiency has been recently shown to cause a severe autoinflammatory syndrome in humans. In this study we characterized the molecular mechanisms whereby interleukin 10 (IL‐10) potentiates IL‐1ra transcription in LPS‐stimulated monocytes and neutrophils. Using chromatin immunoprecipitation, we show that although NF‐κBp65 and NF‐κBp50 proteins accumulate into the nuclei and bind to the IKBα promoter during LPS stimulation, they are not recruited to the κB sites of the IL‐1ra promoter. However, in response to LPS plus IL‐10, which were found to induce chromatin acetylation, recruitment of both NF‐κBp65 and NF‐κBp50 to the IL‐1ra promoter efficiently occurs in a STAT3‐dependent manner. Accordingly, in neutrophils from hyper‐IgE syndrome patients, who carry a nonfunctional STAT3, IL‐10 failed to promote NF‐κBp65 recruitment to the IL‐1ra promoter and consequently to potentiate LPS‐induced IL‐1ra transcription. Altogether our findings uncover a novel mechanism whereby IL‐10‐activated STAT3 modulates IL‐1ra transcription in LPS‐treated phagocytes by making IL‐1ra promoter accessible to readily available nuclear NF‐KB.—Tamassia, N., Castellucci, M., Rossato, M., Gasperini, S., Bosisio, D., Giacomelli, M., Badolato, R., Cassatella, M. A., Bazzoni, F. Uncovering an IL‐10‐dependent NF‐κB recruitment to the IL‐1ra promoter that is impaired in STAT3 functionally defective patients. FASEB J. 24, 1365–1375 (2010). www.fasebj.org

Granulocyte colony-stimulating factor induces the binding of STAT1 and STAT3 to the IFNγ response region within the promoter of the FcγRI/CD64 gene in human neutrophils

Granulocyte colony‐stimulating factor (G‐CSF) has been recently shown to induce the high‐affinity Fc receptor for IgG (FcγRI/CD64) in human polymorphonuclear neutrophils (PMN). To elucidate the molecular mechanisms whereby G‐CSF exerts this effect, we examined whether the cytokine induces the binding of transcription factors to the IFNγ response region (GRR), a well characterized regulatory element in the FCγRI promoter that is responsible for the transcriptional induction of this gene. Using electrophoretic mobility shift assays, we show that in human PMN, G‐CSF activates a GRR‐binding complex which contains members of the signal transducer and activator of transcription (STAT) family of proteins, namely STAT1 and STAT3. In keeping with this result, treatment of neutrophils with G‐CSF led to tyrosine phosphorylation of STAT3, as determined by immunoprecipitation followed by immunoblotting with antiphosphotyrosine antibodies. This is the first demonstration that in human neutrophils, the induction by G‐CSF of FcγRI gene expression may be mediated by the binding of STAT1 and STAT3 to the GRR sequence.

Lipopolysaccharide primes neutrophils for a rapid response to IL‐10

Responsiveness of human neutrophils to IL‐10 was recently shown to be strictly dependent on the levels of IL‐10R1 expression. Activation of signal transducer and activator of transcription 3 (STAT3) phosphorylation and induction of suppressor of cytokine signaling (SOCS)‐3 protein by IL‐10 are in fact negligible in circulating or freshly isolated (“time 0”) neutrophils, but become readily measurable in neutrophils cultured for 4 h in the presence or absence of LPS. In this study, we show that modulation by IL‐10 of LPS‐induced TNF‐α, CXCL8/IL‐8 and IL‐1 receptor antagonist (IL‐1ra) mRNA accumulation in neutrophils already expressing a functional IL‐10R and antigenic SOCS‐3 (i.e. in “4‐h‐cultured” neutrophils) occurs with kinetics that are similar to those observed in “time 0” neutrophils, depends on de novo protein synthesis, but does not require SOCS‐1, SOCS‐3, heme oxygenase and Bcl‐3 induction. By contrast, we show that IL‐10 alone rapidly modulates the expression of TNF‐α, CXCL8/IL‐8 and IL‐1ra mRNA, without any new protein synthesis requirement, if neutrophils have been previously exposed to LPS for at least 4 h. These findings suggest that LPS prepares neutrophils to optimally respond to IL‐10 in terms of rapid gene modulation via mechanisms that, presumably, depend on specific LPS‐induced protein(s).

Impaired cytokine production by neutrophils isolated from patients with AIDS

Objectives:To determine the ability of neutrophils isolated from HIV-seropositive patients to produce proinflammatory cytokines. Design:The in vitro responsiveness of polymorphonuclear neutrophils (PMN) and peripheral blood mononuclear cells (PBMC) to lipopolysaccharide (LPS), used in the presence or absence of interferon (IFN)-γ, was determined in 47 HIV-positive patients with advanced stages of virus infection. Methods:Cytokines in cell-free supernatants were measured by enzyme-linked immunosorbent assay or radioimmunoassay. Results:Cell-free supernatants from PMN isolated from the peripheral blood of HIV-positive patients and stimulated with LPS contained increased amounts of tumour necrosis factor (TNF)-α and interleukin (IL)-8 with respect to supernatants obtained from PMN of normal donors. In contrast, release of IL-1β and IL-1ra (IL-1 receptor antagonist) in response to LPS, or LPS plus IFN-γ, was found to be lower in PMN from HIV-positive patients than in PMN from controls, but was significant only in the case of IL-1ra. Furthermore, the release of IL-12 induced by LPS or LPS plus IFN-γ did not significantly differ between PMN from HIV-positive patients and healthy donors. Concerning PBMC, the production of TNF-α and IL-12 in response to LPS, or LPS plus IFN-γ, was found to be significantly higher in cells isolated from HIV-positive patients, whereas the release of IL-1β was significantly lower. In the case of IL-8, no statistically significant difference was found between PBMC isolated from HIV-positive patients and healthy donors. Conclusions:Collectively, the data suggest that in HIV-positive patients with advanced stages of disease, the ability of PMN to produce specific cytokines in response to LPS is significantly altered. Alterations in this ability might contribute to the evolution of HIV disease.

Impaired natural killer cell functions in patients with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations

Background Gain‐of‐function (GOF) mutations affecting the coiled‐coil domain or the DNA‐binding domain of signal transducer and activator of transcription 1 (STAT1) cause chronic mucocutaneous candidiasis disease. This condition is characterized by fungal and bacterial infections caused by impaired generation of TH17 cells; meanwhile, some patients with chronic mucocutaneous candidiasis disease might also have viral or intracellular pathogen infections. Objective We sought to investigate the effect of STAT1 GOF mutations on the functioning of natural killer (NK) cells. Methods Because STAT1 is involved in the signaling response to several cytokines, we studied NK cell functional activities and STAT1 signaling in 8 patients with STAT1 GOF mutations. Results Functional analysis of NK cells shows a significant impairment of cytolytic and degranulation activities in patients with STAT1 GOF mutations. Moreover, NK cells from these patients display lower production of IFN‐&ggr; in response to IL‐15 and reduced proliferation after stimulation with IL‐2 or IL‐15, suggesting that STAT5 signaling is affected. In addition, signaling studies demonstrate that the increased phosphorylation of STAT1 in response to IFN‐&agr; is associated with detectable activation of STAT1 and increased STAT1 binding to the interferon‐induced protein with tetratricopeptide repeats 1 (IFIT1) promoter in response to IL‐15, whereas STAT5 phosphorylation and DNA binding to IL‐2 receptor &agr; (IL2RA) are reduced or not affected in response to the same cytokine. Conclusion These observations suggest that persistent activation of STAT1 might affect NK cell proliferation and functional activities. Graphical abstract Figure. No Caption available.