Different respiratory syncytial virus and Quillaja saponin formulations induce murine peritoneal cells to express different proinflammatory cytokine profiles

The recognition of a pathogen or a vaccine antigen formulation by cells in the innate immune system leads to production of proinflammatory cytokines, which will determine the ensuing acquired immune response quantitatively and qualitatively. Tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 and IL-6 are the first set of cytokines produced upon such an encounter, which have roles both in protective immunity and immunopathogenesis evident with respiratory syncytial virus (RSV). RSV antigens in different physical adjuvant-vaccine formulations were analysed for their capacity to provoke cultured murine peritoneal cells to produce these three proinflammatory cytokines. RSV immunostimulating complex (ISCOM), i.e. both antigen and adjuvant are incorporated in the same particle, induced high levels of IL-1alpha being of the same magnitude or higher than those of live RSV and lipopolysaccharide (LPS). Live virus and LPS induced higher levels of IL-6 and TNF-alpha than ISCOM and so did non-adjuvanted UV-inactivated RSV but only at high doses. ISCOM-Matrix, i.e. ISCOM without antigens, admixed as a separate entity to inactivated RSV, downregulated or blocked the cytokine response to the inactivated RSV in contrast to ISCOM. Kinetic studies showed that ISCOM induced cytokine production first detected at hours 1, 2, 4 for TNF-alpha, IL-6 and IL-1alpha respectively, which was earlier than for the other antigen formulations containing corresponding doses of antigen and/or Quillaja adjuvant. Peak values for production of TNF-alpha and IL-6 were at 8 h and for IL-1alpha at 72 h following stimulation with ISCOM. The delayed appearance of IL-1alpha may reflect the cell-bound nature of this cytokine.     

Function of GRIM-19, a Mitochondrial Respiratory Chain Complex I Protein, in Innate Immunity

Mitochondria respiratory chain (RC), consisting of five multisubunit complexes, is crucial for cellular energy production, reactive oxygen species generation, and regulation of apoptosis. Recently, a few mitochondrial proteins have been reported to be essential for innate immunity, but the function of mitochondrial RC in innate immunity is largely unknown. By knock-out of GRIM-19, a newly identified subunit protein of mitochondrial complex I, in mice, we found that heterogeneous mice (GRIM-19(+/-)) are prone to spontaneous urinary tract infection, mostly by Staphylococcus saprophyticus. Macrophages derived from these mice have compromised mitochondrial complex I activity and increased reactive oxygen species level. Bacterial infection induces a rapid up-regulation of GRIM-19 and complex I activity in the wild-type macrophages, but both are reduced in the macrophages from GRIM-19(+/-) mice. These cells also have decreased intracellular killing ability against S. saprophyticus. The defects for this probably occur in the fusion of bacteria to lysosome, but not in the bacterial engulfment and macrophage migration. In addition, production of proinflammatory cytokines, such as interleukin (IL)-1, IL-12, IL-6, and interferon (IFN)-γ, induced by both bacterial infection and lipopolysaccharide (LPS) and monodansylcadaverine treatment, is also decreased in the GRIM19(+/-) macrophages. Inhibition of mitochondrial RC activity by inhibitors shows a similar reduction on the cytokine production. Due to low cytokine production, the inflammatory response caused by in vivo bacterial challenge in the bladders of GRIM-19(+/-) mice is compromised. This study provides genetic evidence for a critical role of mitochondrial RC in innate immunity.     

Autocrine deactivation of macrophages in transgenic mice constitutively overexpressing IL-10 under control of the human CD68 promoter

IL-10 plays an essential role in blocking cytokine production by activated macrophages. To analyze the consequences of enforced expression of IL-10 by macrophages on innate and adaptive immune responses, we generated transgenic mice (macIL-10tg mice) expressing an epitope-tagged IL-10 (Flag-IL-10) under control of the human CD68 promoter. Expression of Flag-IL-10 was constitutive and restricted to macrophages, as shown by sorting splenocyte cell populations and intracellular staining for IL-10. Transgenic macrophages displayed suppressed production of TNF-alpha and IL-12 upon stimulation with LPS. When macIL-10tg mice were challenged with LPS, serum levels of proinflammatory cytokines were attenuated compared with controls. Infection with Mycobacterium bovis bacille Calmette-Guérin resulted in approximately 10-fold-higher bacterial loads than in wild-type mice. Normal T and B cell responses were observed in macIL-10tg mice, suggesting that macrophage-specific overexpression of IL-10 predominantly acts in an autocrine/paracrine manner, resulting in chronically deactivated macrophages that manifest an impaired ability to control pathogens.     

Calcium phosphate nanoparticles show an effective activation of the innate immune response in vitro and in vivo after functionalization with flagellin

For subunit vaccines, adjuvants play a key role in shaping the magnitude, persistence and form of targeted antigen-specific immune response. Flagellin is a potent immune activator by bridging innate inflammatory responses and adaptive immunity and an adjuvant candidate for clinical application. Calcium phosphate nanoparticles are efficient carriers for different biomolecules like DNA, RNA, peptides and proteins. Flagellin-functionalized calcium phosphate nanoparticles were prepared and their immunostimulatory effect on the innate immune system, i.e. the cytokine production, was studied. They induced the production of the proinflammatory cytokines IL-8 (Caco-2 cells) and IL-1β (bone marrow-derived macrophages; BMDM) in vitro and IL-6 in vivo after intraperitoneal injection in mice. The immunostimulation was more pronounced than with free flagellin.     

Suppression of innate immunity by acute ethanol administration: a global perspective and a new mechanism beginning with inhibition of signaling through TLR3

Excessive consumption of ethanol (EtOH) suppresses innate immunity, but the mechanisms have not been fully delineated. The present study was conducted to determine whether EtOH suppresses TLR signaling in vivo in mice and to characterize the downstream effects of such suppression. Degradation of IL-1R-associated kinase 1 induced by a TLR3 ligand in peritoneal cells ( approximately 90% macrophages) was suppressed by EtOH. Phosphorylation of p38 kinase in peritoneal macrophages (F4/80(+)) was suppressed, as was nuclear translocation of p-c-Jun and p65 in peritoneal cells. EtOH decreased IL-6 and IL-12 (p40), but did not significantly affect IL-10 in peritoneal lavage fluid or in lysates of peritoneal cells. Changes in cytokine mRNAs (by RNase protection assay) in macrophages isolated by cell sorting or using Ficoll were generally consistent with changes in protein levels in cell lysates and peritoneal lavage fluid. Thus, suppression of TLR signaling and cytokine mRNA occurred in the same cells, and this suppression generally corresponded to changes in i.p. and intracellular cytokine concentrations. DNA microarray analysis revealed the suppression of an IFN-related amplification loop in peritoneal macrophages, associated with decreased expression of numerous innate immune effector genes (including cytokines and a chemokine also suppressed at the protein level). These results indicate that EtOH suppresses innate immunity at least in part by suppressing TLR3 signaling, suppressing an IFN-related amplification loop, and suppressing the induction of a wide range of innate effector molecules in addition to proinflammatory cytokines and chemokines.     

Trichomonas vaginalis inhibits proinflammatory cytokine production in macrophages by suppressing NF-kappaB activation

Activation of NF-kappaB leads to the production of proinflammatory cytokines such as IL-12 and TNF-alpha that are involved in innate and adaptive immunity. We determined whether T. vaginalis-induced inflammatory response in macrophages associated with NF-kappaB. T. vaginalis adhesion led to transient NF-kappaB activation at 6 h but activation declined dramatically by 8 h. Super-shift assays showed that the gel-shifted complexes consisted of p65 (Rel A) and p50 (NF-kappaB1). NF-kappaB activation was accompanied by IkappaB-alpha degradation, and was inhibited by blocking T. vaginalis adhesion, indicating that the early NF-kappaB activation by T. vaginalis depends on IkappaB-alpha degradation. Quantitative real-time RT-PCR analyses revealed that the expression of TNF-alpha and IL-12 mRNA in T. vaginalis-adhesive cells was rapidly suppressed in comparison with LPS stimulation. We also observed that the parasite inhibited the nuclear translocation of NF-kappaB at 8 h, and diminished IL-12 and TNF-alpha production in response to LPS. In addition, inhibition of IkappaB-alpha degradation by MG-132 resulted in apoptosis. These results demonstrate that effects of T. vaginalis on NF-kappaB regulation are critical for cytokine production and the survival of macrophages. We suggest that there exist inhibitory mechanisms induced by T. vaginalis to evade host immunity.     

UVB irradiation suppresses cytokine production and innate cellular immune functions in mice

We examined whether ultraviolet-B (UVB) irradiation (6 kJ/m2) alters cytokine production and other innate immune reactions by murine peritoneal macrophages and peripheral neutrophils. Along with these experiments, serum IgG levels were also assessed. In addition, using scanning electron microscopy (SEM) we observed macrophages that had been exposed to UVB in vitro. Results showed that UVB irradiation: (1) decreased IL-12 production while increasing IL-1alpha secretion from macrophages, but had no effect on IL-1alpha from neutrophils; (2) suppressed phagocytosis of macrophages but not of neutrophils; (3) diminished active oxygen production of macrophages but not of neutrophils; (4) had no effect on serum IgG levels; and (5) caused significant cell destruction of macrophages in vitro. These results suggested: (1) that UVB irradiation could induce characteristic suppression of innate immunity; (2) that innate cellular immunity was more susceptible to the effects of UVB irradiation than humoral immunity.     

B1-B cells are the main antigen presenting cells in CpG-ODN-stimulated peritoneal exudate cells

Peritoneal exudate cells (PEC) have long been used as antigen presenting cells (APC), because they have been considered to contain mainly macrophages. However, it is still unclear specifically which cells of the peritoneal exudate function as APC. Herein, we focused on macrophages and B1-B cells of the PEC and examined their APC function and cytokine production. B1-B cells purified from PEC functioned effectively as APC after CpG-stimulation and mainly produced IL-10. In contrast, macrophages purified from PEC were not able to present incorporated antigens to T cells, despite the production of IL-12 and expression of co-stimulatory molecules after CpG stimulation. These results suggest that previously held ideas regarding the functions of the mixture of cells in the PEC need to re-evaluated. In summary, the antigen presenting function of PEC was mainly attributed to B1-B cells and immunoenhancing cytokine production was dominantly derived from peritoneal macrophages.     

IL-12 and Viral Infections

Interleukin-12 activates natural killer cells and promotes the differentiation of Th1 CD4+ cells; it is a critical factor in viral immunity. IL-12 is secreted by antigen presenting cells including dendritic cells, macrophages and astrocytes, both in tissues and in secondary lymphoid organs. Experimental studies have shown that administration of the cytokine rapidly activates both innate and specific immune responses; this results in enhanced host cellular responses and generally, promotes clearance of virus and host recovery from infection. The observations of many laboratories, studying viral immunity to both RNA and DNA based pathogens, are summarized.     

Intratracheal administration of mitochondrial DNA directly provokes lung inflammation through the TLR9–p38 MAPK pathway

An increasing number of studies have focused on the phenomenon that mitochondrial DNA (mtDNA) activates innate immunity responses. However, the specific role of mtDNA in inflammatory lung disease remains elusive. This study was designed to examine the proinflammatory effects of mtDNA in lungs and to investigate the putative mechanisms. C57BL/6 mice were challenged intratracheally with mtDNA with or without pretreatment with chloroquine. Changes in pulmonary histopathology, cytokine concentrations, and phosphorylation levels of p38 MAPK were assayed at four time points. In in vitro experiments, THP-1 macrophages were pretreated or not pretreated with chloroquine, TLR9 siRNA, p38 MAPK siRNA, or SB203580 and then incubated with mtDNA. The levels of cytokines and p-p38 MAPK were detected by ELISA and Western blot, respectively. The intratracheal administration of mtDNA induced infiltration of inflammatory cells, production of proinflammatory cytokines (including IL-1β, IL-6, and TNF-α), and activation of p38 MAPK. The chloroquine pretreatment resulted in an abatement of mtDNA-induced local lung inflammation. In vitro experiments showed that the exposure of THP-1 macrophages to mtDNA also led to a significant upregulation of IL-1β, IL-6, and TNF-α and the activation of p38 MAPK. And these responses were inhibited either by chloroquine and TLR9 siRNA or by SB203580 and p38 MAPK siRNA pretreatment. The intratracheal administration of mtDNA induced a local inflammatory response in the mouse lung that depended on the interactions of mtDNA with TLR9 and may be correlated with infiltrating macrophages that could be activated by mtDNA exposure via the TLR9–p38 MAPK signal transduction pathway.     

Interleukin-27 Induces a STAT1/3- and NF-κB-dependent Proinflammatory Cytokine Profile in Human Monocytes

IL-27 is a heterodimeric cytokine bridging innate and adaptive immunity by playing a role in the activation of naive T cells and in development of Th1 cells. Additionally, recent evidence supports a role for IL-27 in the activation of monocytic cells. Both pro-inflammatory and anti-inflammatory activities have been attributed to IL-27; however, the role played by IL-27 in the activation of human monocytic cells in terms of cytokine production has not been well described. Our results show that IL-27 is a strong inducer of proinflammatory cytokine and chemokine expression, including enhancement of IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α expression in human primary monocytes. Furthermore, we observed that IL-27-induced cytokine and chemokine production was mediated by STAT1, STAT3, and NF-κB activation. Understanding how IL-27 exerts its effects on monocytic cells will identify important molecular mechanisms in the regulation of immune responses, particularly in the modulation of monocyte activation.     

Enhanced resistance to Gram-positive bacterium and increased susceptibility to bacterial endotoxin in mice sensitized with Propionibacterium acnes: involvement of Toll-like receptor

Mice sensitized with Propionibacterium acnes showed an enhanced resistance against infection with Listeria monocytogenes in contrast to the increased susceptibility to LPS-induced endotoxin shock. The enhanced protection to L. monocytogenes was mediated by activated innate immunity but not by generation of Listeria-specific acquired immunity. After infection with L. monocytogenes, the elimination of bacteria was observed earlier in accordance with a higher level of endogenous cytokine production in P. acnes-sensitized mice than in control mice. Peritoneal cells from P. acnes-sensitized mice produced a larger amount of IL-12p70 and nitric oxide after stimulation with heat-killed L. monocytogenes or peptidoglycan purified from Staphylococcus aureus. RT-PCR analysis showed that the expression of TLR2 but not TLR1, TLR4 nor TLR6 was induced by injection of P. acnes in peritoneal cells. These results indicated that P. acnes-sensitization could induce the activation of innate immunity against L. monocytogenes through increased recognition of bacterial components by TLR2.     

GCN2-Dependent Metabolic Stress Is Essential for Endotoxemic Cytokine Induction and Pathology

Activated inflammatory macrophages can express indoleamine 2,3-dioxygenase (IDO) and thus actively deplete their own tryptophan supply; however, it is not clear how amino acid depletion influences macrophage behavior in inflammatory environments. In this report, we demonstrate that the stress response kinase GCN2 promotes macrophage inflammation and mortality in a mouse model of septicemia. In vitro, enzymatic amino acid consumption enhanced sensitivity of macrophages to the Toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) with significantly increased interleukin 6 (IL-6) production. Tryptophan withdrawal induced the stress response proteins ATF4 and CHOP/GADD153; however, LPS stimulation rapidly enhanced expression of both proteins. Moreover, LPS-driven cytokine production under amino acid-deficient conditions was dependent on GCN2, as GCN2 knockout (GCN2KO) macrophages had a significant reduction of cytokine gene expression after LPS stimulation. To test the in vivo relevance of these findings, monocytic-lineage-specific GCN2KO mice were challenged with a lethal dose of LPS intraperitoneally (i.p.). The GCN2KO mice showed reduced inflammatory responses, with decreased IL-6 and IL-12 expression correlating with significant reduction in animal mortality. Thus, the data show that amino acid depletion stress signals (via GCN2) synergize with proinflammatory signals to potently increase innate immune responsiveness.     

Siglec-9 enhances IL-10 production in macrophages via tyrosine-based motifs

We examined whether Siglec-9 modulates cytokine production in the macrophage cell line RAW264. Cells expressing Siglec-9 produced low levels of tumor necrosis factor (TNF)-α upon stimulation with lipopolysaccharide, peptidoglycan, unmethylated CpG DNA, and double-stranded RNA. On the other hand, interleukin (IL)-10 production was strongly enhanced in Siglec-9-expressing cells. Similar activities were also exhibited by Siglec-5. However, the up-regulation of IL-10 as well as the down-regulation of TNF-α was abrogated when two tyrosine residues in the cytoplasmic tail of Siglec-9 were mutated to phenylalanine. A membrane proximal ITIM mutant of Siglec-9 did not enhance IL-10 production but partly inhibited TNF-α production, indicating diverse regulation mechanisms of TNF-α and IL-10. Siglec-9 also enhanced the production of IL-10 in the human macrophage cell line THP-1. These results demonstrate that Siglec-9 enhances the production of the anti-inflammatory cytokine IL-10 in macrophages.     

IL-15 and macrophage secretory factors facilitate immune activation of neonatal natural killer cells by lipoteichoic acid

Neonates possess a relatively “naive”, yet inducible immune system. Our hypothesis is that upon strategic antigen exposure, cytokine priming and sensitization by accessory cells, natural killer (NK) cells could be activated to become a functional phenotype. We investigated the in vitro stimulation of cord blood (CB) and adult NK cells upon challenge with lipoteichoic acid (LTA), interleukin (IL)-15 and LTA-primed autologous macrophage-conditioned medium, using CD107a and CD69 phenotypes as indicators of activation. We also examined response of CB macrophages to LTA, in terms of P44/42 extracellular signal-regulated kinases (ERK1/2) activation and cytokine secretion. LTA significantly induced secretion of inflammatory cytokines tumor necrotic factor (TNF)-α, IL-6, IL-12 and activated the upstream signal of ERK1/2 phosphorylation in neonatal macrophages. The magnitude of responses to stimulation differed between neonatal and adult NK cells. Co-stimulation with IL-15 was critical for expansion of the CD69 and CD107a NK subpopulations in both neonatal and adult cells, upon a LTA challenge. NK cell activation could be enhanced by LTA-primed autologous macrophages through secretory factors. Our results indicated that neonatal macrophages and NK cells can evoke immunologic responses to a Gram-positive bacterial antigen. The combinatory priming strategy is relevant for development of novel protocols, such as IL-15 treatment, to compensate for the immaturity of the innate immune system in newborns against bacterial infections.     

Neutrophils, dendritic cells and Toxoplasma

Toxoplasma gondii rapidly elicits strong Type 1 cytokine-based immunity. The necessity for this response is well illustrated by the example of IFN-gamma and IL-12 gene knockout mice that rapidly succumb to the effects of acute infection. The parasite itself is skilled at sparking complex interactions in the innate immune system that lead to protective immunity. Neutrophils are one of the first cell types to arrive at the site of infection, and the cells release several proinflammatory cytokines and chemokines in response to Toxoplasma. Dendritic cells are an important source of IL-12 during infection with T. gondii and other microbial pathogens, and they are also specialized for high-level antigen presentation to T lymphocytes. Tachyzoites express at least two types of molecules that trigger innate immune cell cytokine production. One of these involves Toll-like receptor/MyD88 pathways common to many microbial pathogens. The second pathway is less conventional and involves molecular mimicry between a parasite cyclophilin and host CC chemokine receptor 5-binding ligands. Neutrophils, dendritic cells and Toxoplasma work together to elicit the immune response required for host survival. Cytokine and chemokine cross-talk between parasite-triggered neutrophils and dendritic cells results in recruitment, maturation and activation of the latter. Neutrophil-empowered dendritic cells possess properties expected of highly potent antigen presenting cells that drive T helper 1 generation.     

IL-17 production by thymocytes upon CD3 stimulation and costimulation with microbial factors

IL-17 is a potent proinflammatory cytokine produced by activated memory T cells. Recent studies in both human autoimmune diseases and in their animal models have indicated that IL-17 rather than IFN-gamma might be the essential T-cell effector cytokine in the T-cell mediated autoimmune process. Since the thymus has a central role in maintaining T-cell self-tolerance and disturbance of thymic self-tolerance is implied in various autoimmune diseases, we here investigated the capability of murine thymocytes to produce IL-17. Our results indicate that thymocytes are a potent source of IL-17 in response to CD3 stimulation and various microbial immune stimuli and thereby show different patterns in the expression of the proinflammatory cytokines IFN-gamma and IL-17. In addition, strong differences between thymocytes and splenocytes were detected. Altered IL-17 production by thymocytes upon contact with foreign pathogens might be a key regulator in the education of adaptive immunity.     

Antifungal type 1 responses are upregulated in IL-10-deficient mice

C57BL/6 mice are highly resistant to infections caused by Candida albicans and Aspergillus fumigatus. To elucidate the role of IL-10 produced by C57BL/6 mice during these infections, parameters of infection and immunity to it were evaluated in IL-10-deficient and wild-type mice with disseminated or gastrointestinal candidiasis or invasive pulmonary aspergillosis. Unlike parasitic protozoan infection, C. albicans or A. fumigatus infection did not induce significant acute toxicity in IL-10-deficient mice, who, instead, showed reduced fungal burden and fungal-associated inflammatory responses. The increased resistance to infections as compared to wild-type mice was associated with upregulation of innate and acquired antifungal Th1 responses, such as a dramatically higher production of IL-12, nitric oxide (NO) and TNF-alpha as well as IFN-gamma by CD4+ T cells. Pharmacological inhibition of NO production greatly reduced resistance to gastrointestinal candidiasis, thus pointing to the importance of IL-10-dependent NO regulation at mucosal sites in fungal infections. These results are reminiscent of those obtained in genetically susceptible mice, in which IL-10 administration increased, and IL-10 neutralization decreased, susceptibility to C. albicans and A. fumigatus infections. Collectively, these observations indicate that the absence of IL-10 augments innate and acquired antifungal immunity by upregulating type 1 cytokine responses. The resulting protective Th1 responses lead to a prompt reduction of fungal growth, thus preventing tissue destruction and lethal levels of proinflammatory cytokines.     

Induction of dendritic cell maturation by IL-18

IL-18 is a pluripotent proinflammatory cytokine produced primarily by antigen presenting cells involved in numerous aspects of immune regulation most notably on lymphoid cells. The effect of IL-18 stimulation on cells in the myeloid compartment, however, has been poorly studied. Human monocytes did not respond to IL-18. However, the human myelomonocytic cell line KG-1 and monocyte-derived dendritic cells (generated by GM-CSF+IL-4) showed a marked increase in CD83, HLA-DR, and several costimulatory molecules upon stimulation with IL-18. Furthermore, IL-18 decreased pinocytosis of these cells and increased their ability to stimulate alloreactive T cell proliferation, all characteristics of mature dendritic cells. These results suggest that IL-18 is involved in the maturation of myeloid DCs, but not differentiation of monocytes into DCs. The finding that IL-18 is involved in the maturation of dendritic cells is both novel and unexpected and indicates another important role for IL-18 as a key regulator of immune responses.