IFN-gamma-mediated survival enables human neutrophils to produce MCP-1/CCL2 in response to activation by TLR ligands

TLRs are key elements of the pathogen recognition mechanism used by the host immune system. Neutrophils express almost all TLRs, and activation of TLRs, such as TLR2 and TLR4, has been shown to induce the production of proinflammatory cytokines and chemokines, potentially linking innate and adaptive immunity. In the present study, we investigated whether activation of TLRs induces neutrophil production of MCP-1/CCL2, a key mediator involved in the development of adaptive immunity. Activation of neutrophils with LPS, lipoteichoic acid, or N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Ser-[S]-Lys did not induce significant MCP-1 production and release; however, the Th1 cytokine IFN-gamma dramatically up-regulated MCP-1 production in cells activated with each TLR ligand. The majority of MCP-1 was released between 24 and 48 h of culture, indicating that this is a late event. The effect of IFN-gamma appeared to be due to its antiapoptotic effect, but not priming effect, revealing a biological consequence of IFN-gamma-induced neutrophil survival. Although IFN-gamma failed to protect neutrophils from cell death at a higher dose of LPS, the p38 MAPK inhibitor SB203580 dramatically increased MCP-1 release and neutrophil survival at this LPS concentration. Thus, p38 MAPK plays a previously uncharacterized role in neutrophil function. Taken together, our results indicate that human neutrophils produce MCP-1 in a Th1 microenvironment and this neutrophil-derived MCP-1 potentially amplifies the development of Th1 adaptive responses.     

TLR2-mediated activation of neutrophils in response to German cockroach frass

It is becoming increasingly clear that innate immune mediators play a role in regulating adaptive immune responses in asthma pathogenesis. Cockroach exposure is a major risk factor for the development of asthma. In this study we asked whether German cockroach (GC) feces (frass) could initiate an innate immune response. Naive BALB/c mice were challenged with a single intratracheal inhalation of GC frass. Proinflammatory cytokines were significantly increased in the bronchoalveolar lavage fluid at 3 h and were maintained at higher than baseline levels for at least 24 h. Neutrophil migration into the airways was evident as early as 3 h but was maximal between 6 and 24 h postinhalation. The early increase in cytokine expression was independent of TLR2 or TLR4. Newly infiltrated airway neutrophils were responsible for maintaining high levels of cytokines in the airways. Using neutrophils as an early marker of the innate immune response, we show that show that neutrophils isolated from the airways following GC frass inhalation express TLR2 and release cytokines. GC frass directly affected neutrophil cytokine production via TLR2, but not TLR4, as evidenced by the use of TLR-neutralizing Abs and neutrophils from TLR-deficient mice. Activation of cytokine expression occurred via GC frass-induced NF-kappaB translocation and DNA binding. These data show that GC frass contains a TLR2 agonist and, to our knowledge, this is the first report of an allergen directly activating cells of the innate immune system via TLR2 and suggests an important link between innate and adaptive immunity.     

Transient TLR activation restores inflammatory response and ability to control pulmonary bacterial infection in germfree mice

Mammals are colonized by an astronomical number of commensal microorganisms on their environmental exposed surfaces. These symbiotic species build up a complex community that aids their hosts in several physiological activities. We have shown that lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness. The present study investigated whether the germfree state and its hyporesponsive phenotype alter host resistance to an infectious bacterial insult. Experiments performed in germfree mice infected with Klebsiella pneumoniae showed that these animals are drastically susceptible to bacterial infection in an IL-10-dependent manner. In germfree mice, IL-10 restrains proinflammatory mediator production and neutrophil recruitment and favors pathogen growth and dissemination. Germfree mice were resistant to LPS treatment. However, priming of these animals with several TLR agonists recovered their inflammatory responsiveness to sterile injury. LPS pretreatment also rendered germfree mice resistant to pulmonary K. pneumoniae infection, abrogated IL-10 production, and restored TNF-α and CXCL1 production and neutrophil mobilization into lungs of infected germfree mice. This effective inflammatory response mounted by LPS-treated germfree mice resulted in bacterial clearance and enhanced survival upon infection. Therefore, host colonization by indigenous microbiota alters the way the host reacts to environmental infectious stimuli, probably through activation of TLR-dependent pathways. Symbiotic gut colonization enables proper inflammatory response to harmful insults to the host, and increases resilience of the entire mammal-microbiota consortium to environmental pressures.     

Ubiquitous calpains promote both apoptosis and survival signals in response to different cell death stimuli

The mu- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. Here we compared cell death responses and apoptotic or survival signaling pathways in primary mouse embryonic fibroblasts (MEFs) derived from wild type or capn4 knock-out mice which lack both mu- and m-calpain activities. Capn4(-/-) MEFs displayed resistance to puromycin, camptothecin, etoposide, hydrogen peroxide, ultraviolet light, and serum starvation, which was consistent with pro-apoptotic roles for calpain. In contrast, capn4(-/-) MEFs were more susceptible to staurosporine (STS) and tumor necrosis factor alpha-induced cell death, which provided evidence for anti-apoptotic signaling roles for calpain. Bax activation, release of cytochrome c, and activation of caspase-9 and caspase-3 all correlated with the observed cell death responses of wild type or capn4(-/-) MEFs to the various challenges, suggesting that calpain might play distinct roles in transducing different death signals to the mitochondria. There was no evidence that calpain cleaved Bcl-2 family member proteins that regulate mitochondrial membrane permeability including Bcl-2, Bcl-xl, Bad, Bak, Bid, or Bim. However, activation of the phosphatidylinositol 3 (PI3)-kinase/Akt survival signaling pathway was compromised in capn4(-/-) MEFs under all challenges regardless of the cell death outcome, and blocking Akt activation using the PI3-kinase inhibitor LY294002 abolished the protective effect of calpain to STS challenge. We conclude that the anti-apoptotic function of calpain in tumor necrosis factor alpha- and STS-challenged cells relates to a novel role in activating the PI3-kinase/Akt survival pathway.     

Autophagy protein Rubicon mediates phagocytic NADPH oxidase activation in response to microbial infection or TLR stimulation

Phagocytosis and autophagy are two important and related arms of the host's first-line defense against microbial invasion. Rubicon is a RUN domain containing cysteine-rich protein that functions as part of a Beclin-1-Vps34-containing autophagy complex. We report that Rubicon is also an essential, positive regulator of the NADPH oxidase complex. Upon microbial infection or Toll-like-receptor 2 (TLR2) activation, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, facilitating its phagosomal trafficking to induce a burst of reactive oxygen species (ROS) and inflammatory cytokines. Consequently, ectopic expression or depletion of Rubicon profoundly affected ROS, inflammatory cytokine production, and subsequent antimicrobial activity. Rubicon's actions in autophagy and in the NADPH oxidase complex are functionally and genetically separable, indicating that Rubicon functions in two ancient innate immune machineries, autophagy and phagocytosis, depending on the environmental stimulus. Rubicon may thus be pivotal to generating an optimal intracellular immune response against microbial infection.     

Increased interaction between PACT molecules in response to stress signals is required for PKR activation

PKR (protein kinase, RNA activated) is an interferon (IFN)-induced serine-threonine protein kinase and is one of the key mediators in IFN's cellular actions. Although double-stranded (ds) RNA is the most relevant PKR activator during viral infections, PACT acts as a stress-modulated activator of PKR and is an important regulator of PKR dependent signaling pathways in the absence of viral infections. Stress-induced phosphorylation of PACT is essential for PACT's association with PKR leading to PKR activation. PKR activation by PACT leads to phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. In the present study, we have investigated the functional significance of PACT-PACT interaction in mediating PKR activation in response to cellular stress. Our results suggest that enhanced interaction between PACT molecules when PACT is phosphorylated in response to stress signals on serines 246 and 287 is essential for efficient PKR activation. Using a point mutant of PACT that is deficient in PACT-PACT interaction, we demonstrate that PACT-PACT interaction is essential for efficient PKR activation.     

TLR activation of Langerhans cell-like dendritic cells triggers an antiviral immune response

Langerhans cells (LC) are a unique subset of dendritic cells (DC), present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses, we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1-10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types, stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-gamma) and CXCL11 (IFN-gamma-inducible T cell alpha chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally, CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together, our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system.     

Lipid rafts and B-cell activation

The B-cell antigen receptor acts during B-cell activation both to initiate signalling cascades and to transport antigen into the cell for subsequent processing and presentation. Recent evidence indicates that membrane microdomains, termed lipid rafts, have a role in B-cell activation as platforms for B-cell receptor (BCR) signalling and might also act in antigen trafficking. Lipid rafts might facilitate the regulation of the BCR during B-cell development by B-cell co-receptors, and during viral infection. So, lipid rafts seem to be an important new piece of the B-cell signalling puzzle.     

Macrophages and myeloid dendritic cells, but not plasmacytoid dendritic cells, produce IL-10 in response to MyD88- and TRIF-dependent TLR signals, and TLR-independent signals

We have previously reported that mouse plasmacytoid dendritic cells (DC) produce high levels of IL-12p70, whereas bone marrow-derived myeloid DC and splenic DC produce substantially lower levels of this cytokine when activated with the TLR-9 ligand CpG. We now show that in response to CpG stimulation, high levels of IL-10 are secreted by macrophages, intermediate levels by myeloid DC, but no detectable IL-10 is secreted by plasmacytoid DC. MyD88-dependent TLR signals (TLR4, 7, 9 ligation), Toll/IL-1 receptor domain-containing adaptor-dependent TLR signals (TLR3, 4 ligation) as well as non-TLR signals (CD40 ligation) induced macrophages and myeloid DC to produce IL-10 in addition to proinflammatory cytokines. IL-12p70 expression in response to CpG was suppressed by endogenous IL-10 in macrophages, in myeloid DC, and to an even greater extent in splenic CD8alpha(-) and CD8alpha(+) DC. Although plasmacytoid DC did not produce IL-10 upon stimulation, addition of this cytokine exogenously suppressed their production of IL-12, TNF, and IFN-alpha, showing trans but not autocrine regulation of these cytokines by IL-10 in plasmacytoid DC.     

Phospholipase D couples survival and migration signals in stress response of human cancer cells

MDA-MB-231 human breast cancer cells belong to a highly invasive metastatic cell line that depends on phospholipase D (PLD) activity for survival when deprived of serum growth factors. In response to the stress of serum withdrawal, there is a rapid and dramatic increase in PLD activity. Concomitant with increased PLD activity, there was an increase in the ability of MDA-MB-231 cells to both migrate and invade Matrigel. The ability of MDA-MB-231 cells to both migrate and invade Matrigel was dependent on both PLD and mTOR, a downstream target of PLD signals. Serum withdrawal also led to a PLD-dependent increase in the expression of the stress factor, hypoxia-inducible factor-1alpha. These data reveal that PLD survival signals not only prevent apoptosis but also stimulate cell migration and invasion, linking the ability to suppress apoptosis with the ability to metastasize.     

Regulation of lipopolysaccharide-induced B-cell activation: evidence that surface immunoglobulin mediates two independently regulated signals

An antigen-specific B cell response can be induced by low concentrations of haptenated lipopolysaccharide (LPS), whereas high concentrations are inhibitory. Two explanations have been proposed for the latter phenomenon. In the first, specific surface Ig focuses LPS to the B cell membrane, where high local concentrations of the mitogen become paralytic for B cell responses. In the alternative, transmission of an antigen signal at higher concentrations of hapten LPS actively inhibits the development of antibody secreting cells (ASC). In the present paper, the immunosuppressant cyclosporine A (CsA) was used to attempt to distinguish between these two models. Cyclosporine A did not block the inhibitory effects of goat anti-IgM (galphaIgM) on development of ASC induced by LPS and therefore was unsuitable for testing between the two models. However, surprisingly, in the presence but not the absence of CsA, even low concentrations of galphaIgM became inhibitory for LPS-induced B cell proliferation. Thus, a CsA-insensitive signal could inhibit both B cell proliferation and the development of ASC. In contrast, the CsA-sensitive signal induced by sIg required high concentrations of galphaIgM for triggering and enhanced the LPS proliferative response without affecting development of ASC. Evidence is presented that these two signals are regulated independently, suggesting that together they may transmit information about the physical form of an antigen to the B cell.     

Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus

Astrocytes play an important role in initiating and regulating CNS immune responses through the release of proinflammatory cytokines and chemokines. Here we demonstrate that primary astrocytes are capable of recognizing the Gram-positive bacterium Staphylococcus aureus and its cell wall product peptidoglycan (PGN) and respond by producing numerous proinflammatory mediators including interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-1beta (MIP-1beta), MIP-2, and monocyte chemoattractant protein (MCP-1). Astrocytes have recently been shown to express Toll-like receptor 2 (TLR2), a pattern recognition receptor important for recognizing structural components of various Gram-positive bacteria, fungi, and protozoa. However, the functional significance of TLR2 in mediating astrocyte activation remains unknown. Primary astrocytes from TLR2 knockout mice were used to evaluate the role of TLR2 in astrocyte responses to S. aureus and PGN. The results demonstrate that TLR2 is essential for maximal proinflammatory cytokine and chemokine production, but not phagocytosis, in primary astrocytes following S. aureus and PGN exposure. In addition, both stimuli led to a significant increase in TLR2 mRNA expression in wild-type astrocytes as assessed by real-time quantitative RT-PCR. These findings suggest that astrocytes may play a key role in the initial antibacterial immune response in the CNS through engagement of TLR2.     

Impaired naive and memory B-cell responsiveness to TLR9 stimulation in human immunodeficiency virus infection

Toll-like receptor 9 (TLR9) agonists such as unmethylated bacterial CpG DNAs activate B lymphocytes directly, potentially influencing their function and homeostasis. To assess B-cell responsiveness to TLR9 agonists in human immunodeficiency virus (HIV) disease, we examined the ability of naive and memory B cells to proliferation and to increase surface expression of CD80 in response to CpG oligonucleotides (ODN). CpG ODN induced expression of CD80 similarly in B cells from HIV-infected persons and from healthy controls. In contrast, proliferation responses to CpG ODN were markedly impaired in both naive and memory B-cell subsets from HIV-infected persons. Naive B-cell proliferation defects were related to plasma HIV RNA and, among memory B cells, to the frequencies of CD21-negative cells. Importantly, TLR9 mRNA levels were significantly diminished in freshly prepared naive B cells and especially so in memory B cells from HIV-positive viremic donors, suggesting a possible underlying mechanism for the observed functional impairments. Dose-response studies indicated that optimal induction of CD80 expression was achieved with much lower concentrations of CpG ODN than optimal induction of proliferation. We propose that the relatively low threshold of activation that is required for CD80 induction by CpG ODN might explain the preservation of this response in B cells from HIV-infected persons despite diminished TLR9 expression. Impaired responsiveness to TLR9 agonists may contribute to defects in humoral immunity in HIV infection.     

Generalization in response to mate recognition signals

Females usually exhibit strong and unequivocal recognition of conspecific mating signals and reject those of other sympatric heterospecifics. However, most species are allopatric with one another, and the degree to which females recognize mating signals of allopatric species is more varied. Such mating signals are often rejected but are sometimes falsely recognized as conspecific. We studied the dynamics of mate recognition in female túngara frogs (Physalaemus pustulosus) in response to a series of calls that were intermediate between the conspecific and each of five allopatric-heterospecific calls: two that elicited recognition from females in previous studies and three that did not. This study shows that females perceive variation in allopatric mating signals in acontinuous manner with no evidence of perceptual category formation. The strength of recognition is predicted by how different the target stimulus is from the conspecific call within a series of calls. But the differences in recognition responses among call series are not predicted by the similarity of the call series to the conspecific call. The latter result suggests that the strength of recognition of allopatric signals might be influenced by processes of stimulus generalization and past evolutionary history.     

GalR3 activation promotes adult neural stem cell survival in response to a diabetic milieu

Type 2 diabetes impairs adult neurogenesis which could play a role in the CNS complications of this serious disease. The goal of this study was to determine the potential role of galanin in protecting adult neural stem cells (NSCs) from glucolipotoxicity and to analyze whether apoptosis and the unfolded protein response were involved in the galanin‐mediated effect. We also studied the regulation of galanin and its receptor subtypes under diabetes in NSCs in vitro and in the subventricular zone (SVZ) in vivo. The viability of mouse SVZ‐derived NSCs and the involvement of apoptosis (Bcl‐2, cleaved caspase‐3) and unfolded protein response [C/EBP homologous protein (CHOP) Glucose‐regulated protein 78/immunoglobulin heavy‐chain binding protein (GRP78/BiP), spliced X‐box binding protein 1 (XBP1), c‐Jun N‐terminal kinases (JNK) phosphorylation] were assessed in the presence of glucolipotoxic conditions after 24 h. The effect of diabetes on the regulation of galanin and its receptor subtypes was assessed on NSCs in vitro and in SVZ tissues isolated from normal and type 2 diabetes ob/ob mice. We show increased NSC viability following galanin receptor (GalR)3 activation. This protective effect correlated with decreased apoptosis and CHOP levels. We also report how galanin and its receptors are regulated by diabetes in vitro and in vivo. This study shows GalR3‐mediated neuroprotection, supporting a potential future therapeutic development, based on GalR3 activation, for the treatment of brain disorders.