Minja Miettinen

DISTINCT PATTERNS AND KINETICS OF CHEMOKINE PRODUCTION REGULATE DENDRITIC CELL FUNCTION

Dendritic cells (DC) have been showed to both produce and respond to chemokines. To understand how this may impact on DC function, we analyzed the kinetics of chemokine production and responsiveness during DC maturation. After stimulation with LPS, TNF‐α or CD40 ligand, the inflammatory chemokines MIP‐1α, MIP‐1β and IL‐8 were produced rapidly and at high levels, but only for a few hours, while RANTES and MCP‐1 were produced in a sustained fashion. The constitutive chemokines TARC, MDC and PARC were expressed in immature DC and were up‐regulated following maturation, while ELC was produced only at late time points. Activated macrophages produced a similar spectrum of chemokines, but did not produce TARC and ELC. In maturing DC chemokine production had different impact on chemokine receptor function. While CCR1 and CCR5 were down‐regulated by endogenous or exogenous chemokines, CCR7 levels gradually increased in maturing DC and showed a striking resistance to ligand‐induced down‐regulation, explaining how DC can sustain the response to SLC and ELC throughout the maturation process. The time‐ordered production of inflammatory and constitutive chemokines provides DC with the capacity to self‐regulate their migratory behavior as well as to recruit other cells for the afferent and efferent limb of the immune response.

Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response.

Macrophages and dendritic cells (DC) play an essential role in the initiation and maintenance of immune response to pathogens. To analyze early interactions between Mycobacterium tuberculosis (Mtb) and immune cells, human peripheral blood monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were infected with Mtb. Both cells were found to internalize the mycobacteria, resulting in the activation of MDM and maturation of MDDC as reflected by enhanced expression of several surface Ags. After Mtb infection, the proinflammatory cytokines TNF-α, IL-1, and IL-6 were secreted mainly by MDM. As regards the production of IFN-γ-inducing cytokines, IL-12 and IFN-α, was seen almost exclusively from infected MDDC, while IL-18 was secreted preferentially by macrophages. Moreover, Mtb-infected MDM also produce the immunosuppressive cytokine IL-10. Because IL-10 is a potent inhibitor of IL-12 synthesis from activated human mononuclear cells, we assessed the inhibitory potential of this cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12 secretion from Mtb-infected MDM. In line with these findings, supernatants from Mtb-infected MDDC induced IFN-γ production by T cells and enhanced IL-18R expression, whereas supernatants from MDM failed to do that. Neutralization of IFN-α, IL-12, and IL-18 activity in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12 and, to a lesser extent, IFN-α and IL-18 play a significant role in enhancing IFN-γ synthesis by T cells. During Mtb infection, macrophages and DC may have different roles: macrophages secrete proinflammatory cytokines and induce granulomatous inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response.

Lactobacilli and Streptococci Activate NF-κB and STAT Signaling Pathways in Human Macrophages

Gram-positive bacteria induce the production of several cytokines in human leukocytes. The molecular mechanisms involved in Gram-positive bacteria-induced cytokine production have been poorly characterized. In this work we demonstrate that both nonpathogenic Lactobacillus rhamnosus GG and pathogenic Streptococcus pyogenes (group A streptococci) induce NF-κB and STAT DNA-binding activity in human primary macrophages as analyzed by EMSA. NF-κB activation was rapid and was not inhibited by a protein synthesis inhibitor cycloheximide, suggesting that these bacteria could directly activate NF-κB. STAT1, STAT3, and IFN regulatory factor-1 DNA binding was induced by both bacteria with delayed kinetics compared with NF-κB. In addition, streptococci induced the formation of IFN-α-specific transcription factor complex and IFN-stimulated gene factor-3 (ISGF3). STAT1 and STAT3 activation and ISGF3 complex formation were inhibited by cycloheximide or by neutralization with IFN-α/β-specific Abs. Streptococci were more potent than lactobacilli in inducing STAT1, ISGF3, and IFN regulatory factor-1 DNA binding. Accordingly, only streptococci induced IFN-α production. The activation of the IFN-α signaling pathway by streptococci could play a role in the pathogenesis of these bacteria. These results indicate that extracellular Gram-positive bacteria activate transcription factors involved in cytokine signaling by two mechanisms: directly, leading to NF-κB activation, and indirectly via cytokines, leading to STAT activation.

Streptococcus pyogenes and Lactobacillus rhamnosus differentially induce maturation and production of Th1-type cytokines and chemokines in human monocyte-derived dendritic cells

Dendritic cells (DCs) are the most efficient antigen‐presenting cells and thus, have a major role in regulating host immune responses. In the present study, we have analyzed the ability of Gram‐positie, pathogenic Streptococcus pyogenes and nonpathogenic Lactobacillus rhamnosus to induce the maturation of human monocyte‐derived DCs. Stimulation of DCs with S. pyogenes resulted in strong expression of DC costimulatory molecules CD80, CD83, and CD86 accompanied with a T helper cell type 1 (Th1) cytokine and chemokine response. S. pyogenes also induced interleukin (IL)‐2 and IL‐12 production at mRNA and protein levels. In addition, IL‐23 and IL‐27 subunits p40, p19, p28, and EBI3 were induced at mRNA level. In contrast, L. rhamnosus‐stimulated DCs showed only moderate expression of costimulatory molecules and produced low levels of cytokines and chemokines. Furthermore, no production of IL‐2 or IL‐12 family cytokines was detected. Bacteria‐induced DC maturation and especially cytokine and chemokine production were reduced when bacteria were heat‐inactivated. Our results show that human monocyte‐derived DCs respond differently to different Gram‐positive bacteria. Although pathogenic S. pyogenes induced a strong Th1‐type response, stimulation with nonpathogenic L. rhamnosus resulted in development of semi‐mature DCs characterized by moderate expression of costimulatory molecules and low cytokine production.

IFN‐α and IL‐18 synergistically enhance IFN‐γ production in human NK cells: differential regulation of Stat4 activation and IFN‐γ gene expression by IFN‐α and IL‐12

IFN‐γ , a product of NK and T cells, is a key cytokine contributing innate and adaptive immunity. IFN‐γ  production is induced via direct cell‐cell contacts with APC and IFN‐γ ‐producing cells or by cytokines. During microbial infections macrophage‐derived IFN‐α , IL‐12, and IL‐18 enhance IFN‐γ  production and Th1 response. Here we show that IFN‐α  in combination with IL‐18 very efficiently induces IFN‐γ  expression also in primary, nonactivated NK cells and in NK‐92 cell line. Comparison of the kinetics of IFN‐γ  mRNA expression in nonactivated NK cells, NK‐92 cells and activated T cells stimulated with IFN‐α  or IL‐12 revealed that, although both of these cytokines directly up‐regulate IFN‐γ  mRNA expression, its levels remain elevated much longer with IL‐12 stimulation. In both NK cells and T cells, Stat4 is known to be critical in IL‐12 and IFN‐α  signaling. We show that Stat4 activation is transient in cells stimulated with IFN‐α , whereas IL‐12 induces more long‐lasting activation of the transcription factor. This prolonged activation of IFN‐γ  gene by IL‐12 may result in more efficient IFN‐γ  production compared to that of IFN‐α . Our results demonstrate that IFN‐α  and IL‐18 are important innate cytokines in inducing NK cell IFN‐γ  production.

Gene expression profiling during differentiation of human monocytes to macrophages or dendritic cells

Macrophages and dendritic cells (DC) are APC, which regulate innate and adaptive immune responses. Macrophages function locally mainly, maintaining inflammatory responses in tissues, whereas DC take up microbes, mature, and migrate to local lymph nodes to present microbial antigens to naïve T cells to elicit microbe‐specific immune responses. Blood monocytes can be differentiated in vitro to macrophages or DC by GM‐CSF or GM‐CSF + IL‐4, respectively. In the present study, we performed global gene expression analyses using Affymetrix HG‐U133A Gene Chip oligonucleotide arrays during macrophage and DC differentiation. During the differentiation process, 340 and 350 genes were up‐regulated, and 190 and 240 genes were down‐regulated in macrophages and DC, respectively. There were also more that 200 genes, which were expressed differentially in fully differentiated macrophages and DC. Macrophage‐specific genes include, e.g., CD14, CD163, C5R1, and FcγR1A, and several cell surface adhesion molecules, cytokine receptors, WNT5A and its receptor of the Frizzled family FZD2, fibronectin, and FcεR1A were identified as DC‐specific. Our results reveal significant differences in gene expression profiles between macrophages and DC, and these differences can partially explain the functional differences between these two important cell types.

IFN-αβ Released by Mycobacterium tuberculosis-Infected Human Dendritic Cells Induces the Expression of CXCL10: Selective Recruitment of NK and Activated T Cells

We recently reported that dendritic cells (DC) infected with Mycobacterium tuberculosis (Mtb) produce Th1/IFN-γ-inducing cytokines, IFN-αβ and IL-12. In the present article, we show that maturing Mtb-infected DC express high levels of CCR7 and they become responsive to its ligand CCL21. Conversely, CCR5 expression was rapidly lost from the cell surface following Mtb infection. High levels of CCL3 and CCL4 were produced within 8 h after infection, which is likely to account for the observed CCR5 down-modulation on Mtb-infected DC. In addition, Mtb infection stimulated the secretion of CXCL9 and CXCL10. Interestingly, the synthesis of CXCL10 was mainly dependent on the Mtb-induced production of IFN-αβ. Indeed, IFN-αβ neutralization down-regulated CXCL10 expression, whereas the expression of CXCL9 appeared to be unaffected. The chemotactic activity of the Mtb-infected DC supernatants was evaluated by migration assays using activated NK, CD4+, and CD8+ cells that expressed both CCR5 and CXCR3. Mtb-induced expression of CCL3, CCL4, CXCL9, and CXCL10 was involved in the stimulation of NK and T cell migration. In accordance with the data on the IFN-αβ-induced expression of CXCL10, neutralization of IFN-αβ significantly reduced the chemotactic activity of the supernatant from Mtb-infected DC. This indicates that IFN-αβ may modulate the immune response through the expression of CXCL10, which along with CXCL9, CCL3, and CCL4 participates in the recruitment and selective homing of activated/effector cells, which are known to accumulate at the site of Mtb infection and take part in the formation of the granulomas.

Lactobacilli and streptococci induce inflammatory chemokine production in human macrophages that stimulates Th1 cell chemotaxis

Macrophages have a central role in innate‐immune responses to bacteria. In the present work, we show that infection of human macrophages with Gram‐positive pathogenic Streptococcus pyogenes or nonpathogenic Lactobacillus rhamnosus GG enhances mRNA expression of inflammatory chemokine ligands CCL2/monocyte chemoattractant protein‐1 (MCP‐1), CCL3/macrophage‐inflammatory protein‐1α (MIP‐1α), CCL5/regulated on activation, normal T expressed and secreted, CCL7/MCP‐3, CCL19/MIP‐3β, and CCL20/MIP‐3α and CXC chemokine ligands CXCL8/interleukin (IL)‐8, CXCL9/monokine induced by interferon‐γ (IFN‐γ), and CXCL10/IFN‐inducible protein 10. Bacteria‐induced CCL2, CCL7, CXCL9, and CXCL10 mRNA expression was partially dependent on ongoing protein synthesis. The expression of these chemokines and of CCL19 was dependent on bacteria‐induced IFN‐α/β production. CCL19 and CCL20 mRNA expression was up‐regulated by IL‐1β or tumor necrosis factor α (TNF‐α), and in addition, IFN‐α together with TNF‐α further enhanced CCL19 gene expression. Synergy between IFN‐α and TNF‐α was also seen for CXCL9 and CXCL10 mRNA expression. Bacteria‐stimulated macrophage supernatants induced the migration of T helper cell type 1 (Th1) cells, suggesting that in human macrophages, these bacteria can stimulate efficient inflammatory chemokine gene expression including those that recruit Th1 cells to the site of inflammation. Furthermore, L. rhamnosus‐induced Th1 chemokine production could in part explain the proposed antiallergenic properties of this bacterium.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced STAT5 activation and target-gene expression during human monocyte/macrophage differentiation

GM‐CSF signals through JAK2 and STAT5 and stimulates the expression of STAT5 target genes, such as pim‐1 and CIS. Analyzed by EMSA, GM‐CSF stimulation led to much stronger STAT5 DNA‐binding to pim‐1 or CIS GAS elements in primary human monocytes compared with mature macrophages. Similarly, GM‐CSF‐induced expression of pim‐1 and CIS mRNAs was much stronger in monocytes. These differencies were not a result of downregulation of the GM‐CSF receptor system or STAT5 expression, because monocytes and macrophages readily expressed GM‐CSF receptor, JAK2, STAT5A, and STAT5B mRNAs and proteins. Monocytes expressed significant amounts of truncated STAT5 forms that took part in STAT5‐DNA complex formation in GM‐CSF‐stimulated monocytes. This resulted in faster moving STAT5 complexes compared with macrophages in EMSA. Our results demonstrate that STAT5 isoform expression, GM‐CSF‐induced STAT5 activation, and STAT5 target‐gene expression are altered significantly during monocyte/macrophage differentiation.

Live Lactobacillus rhamnosus and Streptococcus pyogenes differentially regulate Toll‐like receptor (TLR) gene expression in human primary macrophages

Macrophages are phagocytes that recognize bacteria and subsequently activate appropriate innate and adaptive immune responses. TLRs are essential in identifying conserved bacterial structures and in initiating and mediating innate immune responses. In this work, we have characterized TLR gene expression in human monocyte‐derived macrophages in response to stimulation with two live Gram‐positive bacteria, a human commensal and probiotic Lactobacillus rhamnosus GG (LGG), and an important human pathogen Streptococcus pyogenes. LGG and S. pyogenes enhanced TLR2 expression in macrophages. LGG and S. pyogenes also required TLR2 for NF‐κB activation. Only pathogenic S. pyogenes was able to up‐regulate TLR3 and TLR7 gene expression. This up‐regulation was dependent on IFN‐α/β, as neutralizing anti‐IFN‐α/β antibodies reduced S. pyogenes‐induced TLR3 and TLR7 mRNA expression. Our results show that despite similarities, TLR responses of macrophages differ for a Gram‐positive probiotic and a pathogen. Our data suggest that macrophages can discriminate between probiotic and pathogenic bacteria by IFN‐mediated TLR gene regulation.