The inflammasome is a multi-protein complex that induces maturation of inflammatory cytokines interleukin (IL)-1β and IL-18 through activation of caspase-1. Several nucleotide binding oligomerization domain-like receptor family members, including NLRP3, recognize unique microbial and danger components and play a central role in inflammasome activation. The NLRP3 inflammasome is critical for maintenance of homeostasis against pathogenic infections. However, inflammasome activation acts as a double-edged sword for various bacterial infections. When the IL-1 family of cytokines is secreted excessively, they cause tissue damage and extensive inflammatory responses that are potentially hazardous for the host. Emerging evidence has shown that diverse bacterial pathogens or their components negatively regulate inflammasome activation to escape the immune response. In this review, we discuss the current knowledge of the roles and regulation of the NLRP3 inflammasome during bacterial infections. Activation and regulation of the NLRP3 inflammasome should be tightly controlled to prevent virulence and pathology during infections. Understanding the roles and regulatory mechanisms of the NLRP3 inflammasome is essential for developing potential treatment approaches against pathogenic infections.
NLRP3 Protein, Human; Bacteria; Infection; Inflammation; Host Defense; Pathology
Among a number of innate receptors, the nucleotide-binding domain leucine-rich repeat containing (NLR) nucleotide oligomerization domain (NOD)-like receptor families are involved in the recognition of cytosolic pathogen- or danger-associated molecules. Activation of these specific sets of receptors leads to the assembly of a multiprotein complex, the inflammasome, leading to the activation of caspase-1 and maturation of the cytokines interleukin (IL)-1β, IL-18, and IL-33. Among NLRs, NLR-related protein 3 (NLRP3) is one of the best-characterized receptors that activates the inflammasome. There is no doubt that NLRP3 inflammasome activation is important for host defense and effective pathogen clearance against fungal, bacterial, and viral infection. In addition, mounting evidence indicates that the NLRP3 inflammasome plays a role in a variety of inflammatory diseases, including gout, atherosclerosis, and type II diabetes, as well as under conditions of cellular stress or injury. Here, we review recent advances in our understanding of the role of the NLRP3 inflammasome in host defense and various inflammatory diseases.
Inflammasomes; Defense mechanisms; Inflammation
Toxoplasma gondii is distributed worldwide and infects most species of warm-blooded animals, including humans. The heavy incidence and severe or lethal damage caused by T. gondii infection clearly indicates the need for the development of a vaccine. To evaluate the protective efficacy of a multiantigenic DNA vaccine expressing GRA7 and ROP1 of T. gondii with or without a plasmid encoding murine interleukin-12 (pIL12), we constructed DNA vaccines using the eukaryotic plasmids pGRA7, pROP1, and pGRA7-ROP1. Mice immunized with pGRA7, pROP1, or pGRA7-ROP1 showed significantly increased serum IgG2a titers; production of gamma interferon (IFN-γ), IL-10, and tumor necrosis factor alpha (TNF-α); in vitro T cell proliferation; and survival, as well as decreased cyst burdens in the brain, compared to mice immunized with either the empty plasmid, pIL12, or vector with pIL12 (vector+pIL12). Moreover, mice immunized with the multiantigenic DNA vaccine pGRA7-ROP1 had higher IgG2a titers, production of IFN-γ and TNF-α, survival time, and cyst reduction rate compared to those of mice vaccinated with either pGRA7 or pROP1 alone. Furthermore, mice immunized with either a pGRA7-ROP1+pIL12 or a single-gene vaccine combined with pIL12 showed greater Th1 immune response and protective efficacy than the single-gene-vaccinated groups. Our data suggest that the multiantigenic DNA antigen pGRA7-ROP1 was more effective in stimulating host protective immune responses than separately injected single antigens, and that IL-12 serves as a good DNA adjuvant.
Autophagy is a housekeeping process that maintains cellular homeostasis through recycling of nutrients and degradation of damaged or aged cytoplasmic constituents. Over the past several years, accumulating evidence has suggested that autophagy can function as an intracellular innate defense pathway in response to infection with a variety of bacteria and viruses. Autophagy plays a role as a specialized immunologic effector and regulates innate immunity to exert antimicrobial defense mechanisms. Numerous bacterial pathogens have developed the ability to invade host cells or to subvert host autophagy to establish a persistent infection. In this review, we have summarized the recent advances in our understanding of the interaction between antibacterial autophagy (xenophagy) and different bacterial pathogens.
autophagy; cytokines; immunity, Innate; infection; reactive oxygen species
There is compelling evidence demonstrating a key role for autophagy in host defense against microbial infections. Induction and regulation of autophagy involves complex pathways including signaling molecules that have widespread roles in cell biological functions. For example, inhibiting mTOR by rapamycin, the most widely used chemical approach to induce autophagy, can also result in immunosupression. Nevertheless, advances in our understanding of autophagy provide a new opportunity to modulate host cellular responses as a potential therapeutic strategy to combat microbial infections in humans.
Studies over the past decade have helped to decipher molecular networks dependent on Toll-like receptor (TLR) signaling, in mycobacteria-infected macrophages. Stimulation of TLRs by mycobacteria and their antigenic components rapidly induces intracellular signaling cascades involved in the activation of nuclear factor-κB and mitogen-activated protein kinase pathways, which play important roles in orchestrating proinflammatory responses and innate defense through generation of a variety of antimicrobial effector molecules. Recent studies have provided evidence that mycobacterial TLR-signaling cross talks with other intracellular antimicrobial innate pathways, the autophagy process and functional vitamin D receptor (VDR) signaling. In this article we describe recent advances in the recognition, responses, and regulation of mycobacterial signaling through TLRs.
mycobacteria; vitamin D; autophagy; antimicrobial peptides; innate immunity
Apoptosis is thought to play a role in host defenses against intracellular pathogens, including Mycobacterium tuberculosis (Mtb), by preventing the release of intracellular components and the spread of mycobacterial infection. This study aims to investigate the role of endoplasmic reticulum (ER) stress mediated apoptosis in mycobacteria infected macrophages.
Here, we demonstrate that ER stress-induced apoptosis is associated with Mtb H37Rv-induced cell death of Raw264.7 murine macrophages. We have shown that Mtb H37Rv induced apoptosis are involved in activation of caspase-12, which resides on the cytoplasmic district of the ER. Mtb infection increase levels of other ER stress indicators in a time-dependent manner. Phosphorylation of eIF2α was decreased gradually after Mtb H37Rv infection signifying that Mtb H37Rv infection may affect eIF2α phosphorylation in an attempt to survive within macrophages. Interestingly, the survival of mycobacteria in macrophages was enhanced by silencing CHOP expression. In contrast, survival rate of mycobacteria was reduced by phosphorylation of the eIF2α. Futhermore, the levels of ROS, NO or CHOP expression were significantly increased by live Mtb H37Rv compared to heat-killed Mtb H37Rv indicating that live Mtb H37Rv could induce ER stress response.
These findings indicate that eIF2α/CHOP pathway may influence intracellular survival of Mtb H37Rv in macrophages and only live Mtb H37Rv can induce ER stress response. The data support the ER stress pathway plays an important role in the pathogenesis and persistence of mycobacteria.
Antimicrobial peptides/proteins are ancient and naturallyoccurring antibiotics in innate immune responses in a variety of organisms. Additionally, these peptides have been recognized as important signaling molecules in regulation of both innate and adaptive immunity. During mycobacterial infection, antimicrobial peptides including cathelicidin, defensin, and hepcidin have antimicrobial activities against mycobacteria, making them promising candidates for future drug development. Additionally, antimicrobial peptides act as immunomodulators in infectious and inflammatory conditions. Multiple crucial functions of cathelicidins in antimycobacterial immune defense have been characterized not only in terms of direct killing of mycobacteria but also as innate immune regulators, i.e., in secretion of cytokines and chemokines, and mediating autophagy activation. Defensin families are also important during mycobacterial infection and contribute to antimycobacterial defense and inhibition of mycobacterial growth both in vitro and in vivo. Hepcidin, although its role in mycobacterial infection has not yet been characterized, exerts antimycobacterial effects in activated macrophages. The present review focuses on recent efforts to elucidate the roles of host defense peptides in innate immunity to mycobacteria.
Antimicrobial peptides; Innate Immunity; Mycobacteria
Mycobacterium tuberculosis (Mtb) heparin binding hemagglutinin (HBHA) is an Ag known to evoke effective host immune responses during tuberculosis infection. However, the molecular basis of the host immune response to HBHA has not been fully characterized. In this study, we examined the molecular mechanisms by which HBHA can induce the expression of proinflammatory cytokines in macrophages.
HBHA-induced mRNA and protein levels of proinflammatory cytokines were determined in bone marrow-derived macrophages (BMDMs) using RT-PCR and ELISA analysis. The roles of intracellular signaling pathways for NF-κB, PI3-K/Akt, and MAPKs were investigated in macrophage proinflammatory responses after stimulation with HBHA.
HBHA robustly activated the expression of mRNA and protein of both TNF-α and IL-6, and induced phosphorylation of NF-κB, Akt, and MAPKs in BMDMs. Both TNF-α and IL-6 production by HBHA was regulated by the NF-κB, PI3-K, and MAPK pathways. Furthermore, PI3-K activity was required for the HBHA-induced activation of ERK1/2 and p38 MAPK, but not JNK, pathways.
These data suggest that mycobacterial HBHA significantly induces proinflammatory responses through crosstalk between the PI3-K and MAPK pathways in macrophages.
Mycobacterium tuberculosis; Heparin-binding hemagglutinin (HBHA); Macrophages; MAPK; PI3-K; NF-κB
In human monocytes, Toll-like receptor (TLR) 2/1 activation leads to vitamin D3-dependent antimycobacterial activities, but the molecular mechanisms by which TLR2/1 stimulation induces antimicrobial activities against mycobacteria remain unclear. Here we show that TLR2/1/CD14 stimulation by mycobacterial lipoprotein LpqH can robustly activate antibacterial autophagy through vitamin D receptor signalling activation and cathelicidin induction. We found that CCAAT/enhancer-binding protein (C/EBP)-β-dependent induction of 25-hydroxycholecalciferol-1α-hydroxylase (Cyp27b1) hydroxylase was critical for LpqH-induced cathelicidin expression and autophagy. In addition, increases in intracellular calcium following AMP-activated protein kinase (AMPK) activation played a crucial role in LpqH-induced autophagy. Moreover, AMPK-dependent p38 mitogen-activated protein kinase (MAPK) activation was required for LpqH-induced Cyp27b1 expression and autophagy activation. Collectively, these data suggest that TLR2/1/CD14-Ca2+-AMPK-p38 MAPK pathways contribute to C/EBP-β-dependent expression of Cyp27b1 and cathelicidin, which played an essential role in LpqH-induced autophagy. Furthermore, these results establish a previously uncharacterized signalling pathway of antimycobacterial host defence through a functional link of TLR2/1/CD14-dependent sensing to the induction of autophagy.
Membrane lipid rafts are enriched in cholesterol and play an important role as signalling platforms. However, the roles of lipid rafts and associated signalling molecules in the innate immune responses to mycobacteria remain unknown. Here we show that stimulation with Mycobacterium tuberculosis 19 kDa lipoprotein, a TLR2/1 agonist, results in translocation of TLR2 to lipid rafts, coalescence of lipid rafts and production of reactive oxygen species (ROS) that drive pro-inflammatory responses. Disruption of lipid raft organization markedly reduced lipoprotein-induced ROS and inflammatory responses. Remarkably, the atypical protein kinase C (PKC) ζ was specifically recruited into detergent-resistant membrane fractions and associated with TLR2. PKCζ activity was critical for lipoprotein-dependent ROS generation, raft coalescence and the pro-inflammatory responses by macrophages. Moreover, lipid raft organization was required for 19 kDa mediated PKCζ activation. These results demonstrate that TLR2 trafficking and raft coalescence play an essential role for the initiation of lipoprotein-induced innate immune responses via TLR2 and ROS signalling. In addition, PKCζ targets to lipid rafts and may act as a critical adaptor molecule to regulate lipid raft dynamics during TLR2 signalling.
Microglial cells are activated during excitotoxin-induced neurodegeneration. However, the in vivo role of microglia activation in neurodegeneration has not yet been fully elucidated. To this end, we used Ikkβ conditional knockout mice (LysM-Cre/IkkβF/F) in which the Ikkβ gene is specifically deleted in cells of myeloid lineage, including microglia, in the CNS. This deletion reduced IκB kinase (IKK) activity in cultured primary microglia by up to 40% compared with wild-type (IkkβF/F), and lipopolysaccharide-induced proinflammatory gene expression was also compromised. Kainic acid (KA)-induced hippocampal neuronal cell death was reduced by 30% in LysM-Cre/IkkβF/F mice compared with wild-type mice. Reduced neuronal cell death was accompanied by decreased KA-induced glial cell activation and subsequent expression of proinflammatory genes such as tumour necrosis factor (TNF)-α and interleukin (IL)-1β. Similarly, neurons in organotypic hippocampal slice cultures (OHSCs) from LysM-Cre/IkkβF/F mouse brain were less susceptible to KA-induced excitotoxicity compared with wild-type OHSCs, due in part to decreased TNF-α and IL-1β expression. Based on these data, we concluded that IKK/nuclear factor-κB dependent microglia activation contributes to KA-induced hippocampal neuronal cell death in vivo through induction of inflammatory mediators.
excitotoxicity; hippocampus; IKKβ; kainic acid; microglia
Little information is available the role of Nitric Oxide (NO) in host defenses during human tuberculosis (TB) infection. We investigated the modulating factor(s) affecting NO synthase (iNOS) induction in human macrophages.
Both iNOS mRNA and protein that regulate the growth of mycobacteria were determined using reverase transcriptase-polymerase chain reaction and western blot analysis. The upstream signaling pathways were further investigated using iNOS specific inhibitors.
Here we show that combined treatment with 1,25-dihydroxyvitamin D3 (1,25-D3) and Interferon (IFN)-γ synergistically enhanced NO synthesis and iNOS expression induced by Mycobacterium tuberculosis (MTB) or by its purified protein derivatives in human monocyte-derived macrophages. Both the nuclear factor-κB and MEK1-ERK1/2 pathways were indispensable in the induction of iNOS expression, as shown in toll like receptor 2 stimulation. Further, the combined treatment with 1,25-D3 and IFN-γ was more potent than either agent alone in the inhibition of intracellular MTB growth. Notably, this enhanced effect was not explained by increased expression of cathelicidin, a known antimycobacterial effector of 1,25-D3.
These data support a key role of NO in host defenses against TB and identify novel modulating factors for iNOS induction in human macrophages.
monocytes/macrophages; nitric oxide; human; bacterial; TLR
We present two cases of Wiskott-Aldrich syndrome (WAS), in which nonsense mutations in the WASP gene were corrected phenotypically as well as genotypically by unrelated cord blood stem cell transplantation (CBSCT). Two male patients were diagnosed with WAS at the age of 5-month and 3-month and each received unrelated CBSCT at 16-month and 20-month of age, respectively. The infused cord blood (CB) units had 4/6 and 5/6 HLA matches and the infusion doses of total nucleated cells (TNC) and CD34+ cells were 6.24×107/kg and 5.08×107/kg for TNC and 1.33×105/kg and 4.8×105/kg for CD34+ cells, for UPN1 and UPN2, respectively. Complete donor cell chimerism was documented by variable number tandem repeat (VNTR) with neutrophil engraftment on days 31 and 13 and platelets on days 58 and 50, respectively. Immunologic reconstitution demonstrated that CBSCT resulted in consistent and stable T-, B-, and NK-cell development. Flow cytometric analysis for immunologic markers and sequence analysis of the WASP gene mutation revealed a normal pattern after CBSCT. These cases demonstrate that CBs can be an important source of stem cells for the phenotypical and genotypical correction of genetic diseases such as WAS.
Wiskott-Aldrich Syndrome; WASP; Unrelated Cord Blood Stem Cell Transplantation
Although tuberculosis poses a significant health threat to the global population, it is a challenge to develop new and effective therapeutic strategies. Nitric oxide (NO) and inducible NO synthase (iNOS) are important in innate immune responses to various intracellular bacterial infections, including mycobacterial infections. It is generally recognized that reactive nitrogen intermediates play an effective role in host defense mechanisms against tuberculosis. In a murine model of tuberculosis, NO plays a crucial role in antimycobacterial activity; however, it is controversial whether NO is critically involved in host defense against Mycobacterium tuberculosis in humans. Here, we review the roles of NO in host defense against murine and human tuberculosis. We also discuss the specific roles of NO in the central nervous system and lung epithelial cells during mycobacterial infection. A greater understanding of these defense mechanisms in human tuberculosis will aid in the development of new strategies for the treatment of disease.
nitric oxide; mycobacteria; macrophages; host defense
Mammalian 2-Cys peroxiredoxin II (Prx II) is a cellular peroxidase that eliminates endogenous H2O2. The involvement of Prx II in the regulation of lipopolysaccharide (LPS) signaling is poorly understood. In this report, we show that LPS induces substantially enhanced inflammatory events, which include the signaling molecules nuclear factor κB and mitogen-activated protein kinase (MAPK), in Prx II–deficient macrophages. This effect of LPS was mediated by the robust up-regulation of the reactive oxygen species (ROS)–generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and the phosphorylation of p47phox. Furthermore, challenge with LPS induced greater sensitivity to LPS-induced lethal shock in Prx II–deficient mice than in wild-type mice. Intravenous injection of Prx II–deficient mice with the adenovirus-encoding Prx II gene significantly rescued mice from LPS-induced lethal shock as compared with the injection of a control virus. The administration of catalase mimicked the reversal effects of Prx II on LPS-induced inflammatory responses in Prx II–deficient cells, which suggests that intracellular H2O2 is attributable, at least in part, to the enhanced sensitivity to LPS. These results indicate that Prx II is an essential negative regulator of LPS-induced inflammatory signaling through modulation of ROS synthesis via NADPH oxidase activities and, therefore, is crucial for the prevention of excessive host responses to microbial products.
Identification and characterization of serologically active mycobacterial antigens are prerequisites for the development of diagnostic reagents. We examined the humoral immune responses of active tuberculosis (TB) patients against Triton-soluble proteins extracted from Mycobacterium tuberculosis by immunoblotting. A 29-kDa protein reacted with immunoglobulin M (IgM) in the pooled sera of the patients, and its N-terminal amino acid sequence matched that of the heparin-binding hemagglutinin (HBHA). Recombinant full-length HBHA was expressed in Escherichia coli (rEC-HBHA) and M. smegmatis (rMS-HBHA). In immunoblot analysis, the IgM antibodies of the TB patients reacted strongly with rMS-HBHA but not with rEC-HBHA, whereas the IgG antibodies of these patients reacted weakly with both recombinant HBHA proteins. In enzyme-linked immunosorbent assay analysis using rMS-HBHA and 85B as antigens, the mean levels and sensitivities of the anti-HBHA IgM antibodies of the TB patients were significantly higher than those of the anti-antigen 85B IgM antibodies, while the IgG antibodies showed the opposite results. Of interest in this respect, the pooled sera from the TB patients that contained anti-HBHA IgM antibodies neutralized the entry of M. tuberculosis into epithelial cells. These findings suggest that IgM antibody to HBHA may play a role in protection against extrapulmonary dissemination.
Although the 38-kDa glycolipoprotein of Mycobacterium tuberculosis H37Rv is known to evoke prominent cellular and humoral immune responses in human tuberculosis (TB), little information is known about intracellular regulatory mechanisms involved in 38-kDa antigen (Ag)-induced host responses. In this study, we found that purified 38-kDa glycolipoprotein activates mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase 1/2 [ERK1/2] and p38) and induces tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in human monocytes. When the 38-kDa Ag was applied to monocytes from TB patients and healthy controls, the activation of ERK1/2 and p38 MAPK and the subsequent cytokine secretion were greater in the monocytes from the active pulmonary TB patients than in monocytes from the healthy controls. Additionally, neutralizing antibodies for Toll-like receptor 2 (TLR2) or TLR4 significantly reduced the ERK1/2 and p38 activation induced by the 38-kDa protein when the antibodies were applied to HEK293 cells overexpressing TLR2 or TLR4 as well as human primary monocytes. Furthermore, the inhibition of TLR2 significantly, and that of TLR4 partially, decreased the 38-kDa Ag-induced secretion of TNF-α and IL-6 in human monocytes. The intact protein moieties of the 38-kDa protein were responsible for biologic activities by this Ag. These data collectively demonstrate that the 38-kDa glycolipoprotein, acting through both TLR2 and TLR4, induces the activation of the ERK1/2 and p38 MAPK pathways, which in turn play an essential role in TNF-α and IL-6 expression during mycobacterial infection.
We examined the distribution of single nucleotide polymorphisms (SNPs) in nitric oxide synthase 2A, monocyte chemoattractant protein–1 (MCP-1), regulated on activation, normal T cell expressed and secreted, and macrophage inflammatory protein–1α genes in tuberculosis patients and healthy controls from Mexico. The odds of developing tuberculosis were 2.3- and 5.4-fold higher in carriers of MCP-1 genotypes AG and GG than in homozygous AA. Cases of homozygous GG had the highest plasma levels of MCP-1 and the lowest plasma levels of IL-12p40, and these values were negatively correlated. Furthermore, stimulation of monocytes from healthy carriers of the genotype GG with Mycobacterium tuberculosis antigens yielded higher MCP-1 and lower IL-12p40 concentrations than parallel experiments with monocytes from homozygous AA. Addition of anti–MCP-1 increased IL-12p40 levels in cultures of M. tuberculosis–stimulated monocytes from homozygous GG, and addition of exogenous MCP-1 reduced IL-12p40 production by M. tuberculosis–stimulated monocytes from homozygous AA. Furthermore, we could replicate our results in Korean subjects, in whom the odds of developing tuberculosis were 2.8- and 6.9-fold higher in carriers of MCP-1 genotypes AG and GG than in homozygous AA. Our findings suggest that persons bearing the MCP-1 genotype GG produce high concentrations of MCP-1, which inhibits production of IL-12p40 in response to M. tuberculosis and increases the likelihood that M. tuberculosis infection will progress to active pulmonary tuberculosis.
Lymphocyte function-associated antigen (LFA)-1 clustering, which is needed for high avidity binding to intercellular adhesion molecule (ICAM)-1 and -2, regulates T cell motility and T cell–antigen-presenting cell (APC) conjugation. In this study, down-regulation of SKAP-55 by small interfering RNAs (siRNAs) identified an essential role for this adaptor molecule in the T cell receptor (TCR)–mediated ”inside-out signaling” that is needed for LFA-1 clustering and T cell–APC conjugation. In contrast, down-regulation of SKAP-55 had no effect on TCR–CD3 clustering. Furthermore, the expression of the related protein SKAP-55R failed to compensate for the loss of SKAP-55 in LFA-1 clustering, indicating that SKAP-55 has a unique function that cannot be replaced by this closely related protein. Our findings therefore indicate that SKAP-55, unlike SKAP-55R, is specifically tailored as an essential component of the inside-out signaling events that couple the TCR to LFA-1 clustering and T cell–APC conjugation.
X-linked severe combined immunodeficiency (X-SCID) is a rare, life-threatening immune disorder, caused by mutations in the γc chain gene, which encodes an essential component of the cytokine receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21. A 13-month-old boy with recurrent infections who had reduced serum immunoglobulin levels and decreased numbers of CD3, CD16/56 cells was evaluated for γc chain gene mutation and protein expression. The patient had a C-to-T point mutation at nucleotide position 690, one of the hot spots, resulting in a single amino acid substitution of cysteine for arginine (R226C), as determined by direct sequencing and PCR-RFLP. The patient's mother was a heterozygous carrier. Percutaneous umbilical cord blood sampling was performed at the 6-month of gestation in a subsequent pregnancy. As the immunophenotype of the fetus showed an identical pattern, the pregnancy was terminated and genetic analysis of the abortus confirmed recurrence. This is the first report of the molecular diagnosis of X-SCID in Korea. Genetic analysis of the γc chain gene is useful for definite diagnosis and genetic counseling for X-SCID.
Severe Combined Immunodeficiency; Genetic Diseases, X-linked; Mutation; Genetic Counseling; Korea
Although Mycobacterium marinum is closely related to Mycobacterium tuberculosis H37Rv genomically, the clinical outcome in humans is quite different for M. marinum and M. tuberculosis infections. We investigated possible factors in the host macrophages for determining differential pathological responses to M. tuberculosis and M. marinum using an in vitro model of mycobacterial infection. Using suppression-subtractive hybridization, we identified 12 differentially expressed genes in the human monocytic cell line U937 infected with M. tuberculosis and M. marinum. Of those genes, the most frequently recovered transcript encoded interleukin-8 (IL-8). Northern hybridization revealed that IL-8 mRNA was highly upregulated in M. tuberculosis-infected U937 cells compared with M. marinum-infected cells. In addition, enzyme-linked immunosorbent assay showed that IL-8 protein secretion was significantly elevated in M. tuberculosis-infected U937 cells, human primary monocytes, and monocyte-derived macrophages compared with that in M. marinum-infected cells. The depressed IL-8 expression was unique in M. marinum-infected cells compared with cells infected with other strains of mycobacteria, including M. tuberculosis H37Ra, Mycobacterium bovis BCG, or Mycobacterium smegmatis. When the expression of NF-κB was assessed in mycobacterium-infected U937 cells, IκBα proteins were significantly degraded in M. tuberculosis-infected cells compared with M. marinum-infected cells. Collectively, these results suggest that differential IL-8 expression in human macrophages infected with M. tuberculosis and M. marinum may be critically associated with distinct host responses in tuberculosis. Additionally, our data indicate that differential signal transduction pathways may underlie the distinct patterns of IL-8 secretion in cells infected by the two mycobacteria.
X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency disorder, caused by mutations of the gene encoding CD40 ligand (CD40L; CD154). We report the clinical manifestations and mutational analysis of the CD40L gene observed in a male patient from a XHIM family. Having hypogammaglobulinemia and elevated IgM, the 3-yr-old boy exhibited the characteristic clinical features of XHIM. The patient suffered from frequent respiratory infections, and chronic enteritis caused by Cryptosporidium parvum. In addition, a lymph node biopsy and a culture from this sample revealed C. neoformans infection. Activated lymphocytes from the patient failed to express CD40L on their surface as assessed by flow cytometry and a missence mutation (W140R) was found at the XHIM hotspot in his CD40L cDNA to confirm the diagnosis. Genetic analysis of the mother and sister showed a heterozygote pattern, indicating carrier status. To our knowledge, this is the first report on the molecular diagnosis of an XHIM patient in Korea.
The secreted 30-kDa antigen (Ag) of Mycobacterium tuberculosis directly stimulates Th1-type protective cytokine responses in healthy tuberculin reactors but not in patients with active tuberculosis (TB). To examine the cytokine profiles attributable to Th1 suppression associated with active TB, interleukin-12 (IL-12), IL-18, and IL-10 production in response to a 30- or 32-kDa Ag in 16 patients with active pulmonary TB and 24 healthy controls was investigated by enzyme-linked immunosorbent assay. In TB patients, production of IL-12 p40, as well as gamma interferon (IFN-γ), by 30- or 32-kDa Ag-stimulated peripheral blood mononuclear cells (PBMC) was significantly decreased compared with that in healthy tuberculin reactors. There were no significant differences in IL-18 production between patients and controls early during stimulation (16 h). However, PBMC from patients showed significantly enhanced IL-18 proteins after 96 h of stimulation. Similarly, higher IL-10 production was observed in the TB patients than in healthy tuberculin reactors. After 2 months of anti-TB therapy, the mean IFN-γ and IL-12 p40 production and the mean blastogenic responses were significantly increased in PBMC in the 10 TB patients who were followed up. Our findings provide evidence that depressed IL-12 in response to the 30- or 32-kDa Ag is involved in the immunopathogenesis of human active pulmonary TB.