The Role of PI3K/AKT Pathway and NADPH Oxidase 4 in Host ROS Manipulation by Toxoplasma gondii

Dendritic cell is one of the first innate immune cell to encounter T. gondii after the parasite crosses the host intestinal epithelium. T. gondii requires intact DC as a carrier to infiltrate into host central nervous system (CNS) without being detected or eliminated by host defense system. The mechanism by which T. gondii avoids innate immune defense of host cell, especially in the dendritic cell is unknown. Therefore, we examined the role of host PI3K/AKT signaling pathway activation by T. gondii in dendritic cell. T. gondii infection or T. gondii excretory/secretory antigen (TgESA) treatment to the murine dendritic cell line DC2.4 induced AKT phosphorylation, and treatment of PI3K inhibitors effectively suppressed the T. gondii proliferation but had no effect on infection rate or invasion rate. Furthermore, it is found that T. gondii or TgESA can reduce H2O2-induced intracellular reactive oxygen species (ROS) as well as host endogenous ROS via PI3K/AKT pathway activation. While searching for the main source of the ROS, we found that NADPH oxidase 4 (NOX4) expression was controlled by T. gondii infection or TgESA treatment, which is in correlation with previous observation of the ROS reduction by identical treatments. These findings suggest that the manipulation of the host PI3K/AKT signaling pathway and NOX4 expression is an essential mechanism for the down-regulation of ROS, and therefore, for the survival and the proliferation of T. gondii.

4-Hydroxybenzaldehyde Restricts the Intracellular Growth of Toxoplasma gondii by Inducing SIRT1-Mediated Autophagy in Macrophages

Toxoplasma gondii is an intracellular protozoan parasite that infects approximately one third of the human popu- lation worldwide. Considering the toxicity and side effects of anti-toxoplasma medications, it is important to develop effec- tive drug alternatives with fewer and less severe off-target effects. In this study, we found that 4-hydroxybenzaldehyde (4- HBA) induced autophagy and the expression of NAD-dependent protein deacetylase sirtuin-1 (SIRT1) in primary murine bone marrow-derived macrophages (BMDMs). Interestingly, treatment of BMDMs with 4-HBA significantly reduced the number of macrophages infected with T. gondii and the proliferation of T. gondii in infected cells. This effect was impaired by pretreating the macrophages with 3-methyladenine or wortmannin (selective autophagy inhibitors) or with sirtinol or EX527 (SIRT1 inhibitors). Moreover, we found that pharmacological inhibition of SIRT1 prevented 4-HBA-mediated expres- sion of LC3-phosphatidylethanolamine conjugate (LC3-II) and the colocalization of T. gondii parasitophorous vacuoles with autophagosomes in BMDMs. These data suggest that 4-HBA promotes antiparasitic host responses by activating SIRT1- mediated autophagy, and 4-HBA might be a promising therapeutic alternative for the treatment of toxoplasmosis.

Dipenyleneiodonium Induces Growth Inhibition of Toxoplasma gondii through ROS Induction in ARPE-19 Cells

Based on the reactive oxygen species (ROS) regulatory properties of diphenyleneiodonium (DPI), we investigated the effects of DPI on host-infected T. gondii proliferation and determined specific concentration that inhibit the intracellular parasite growth but without severe toxic effect on human retinal pigment epithelial (ARPE-19) cells. As a result, it is observed that host superoxide, mitochondria superoxide and H2O2 levels can be increased by DPI, significantly, followed by suppression of T. gondii infection and proliferation. The involvement of ROS in anti-parasitic effect of DPI was confirmed by finding that DPI effect on T. gondii can be reversed by ROS scavengers, N-acetyl-L-cysteine and ascorbic acid. These results suggest that, in ARPE-19 cell, DPI can enhance host ROS generation to prevent T. gondii growth. Our study showed DPI is capable of suppressing T. gondii growth in host cells while minimizing the un-favorite side-effect to host cell. These results imply that DPI as a promising candidate material for novel drug development that can ameliorate toxoplasmosis based on ROS regulation.

Fasciola hepatica: Infection Status of Freshwater Snails Collected from Gangwon-do (Province), Korea

Fasciola hepatica is a trematode that causes zoonosis, mainly in cattle and sheep, and occasionally in humans. Few recent studies have determined the infection status of this fluke in Korea. In August 2015, we collected 402 samples of freshwater snails at Hoenggye-ri (upper stream) and Suha-ri (lower stream) of Song-cheon (stream) in Daegwalnyeong-myeon, Pyeongchang-gun in Gangwon-do (Province) near many large cattle or sheep farms. F. hepatica infection was determined using PCR on the nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 402 samples, F. hepatica 1TS-2 marker was detected in 6 freshwater snails; thus, the overall prevalence in freshwater snails was 1.5%. The prevalence varied between collection areas, ranging from 0.0% at Hoenggye-ri to 2.9% at Suha-ri. However, F. gigantica ITS-2 was not detected in the 6 F. hepatica-positive samples by PCR. The nucleotide sequences of the 6 F. hepatica ITS-2 PCR-positive samples were 99.4% identical to the F. hepatica ITS-2 sequences in GenBank, whereas they were 98.4% similar to F. gigantica ITS-2 sequences. These results indicated that the prevalence of F. hepatica in snail intermediate hosts was 1.5% in Gangwon-do, Korea; however the prevalence varied between collection areas. These results may help us to understand F. hepatica infection status in natural environments.

IL-12 and IL-23 Production in Toxoplasma gondii- or LPS-Treated Jurkat T Cells via PI3K and MAPK Signaling Pathways

IL-12 and IL-23 are closely related in structure, and have been shown to play crucial roles in regulation of immune responses. However, little is known about the regulation of these cytokines in T cells. Here, we investigated the roles of PI3K and MAPK pathways in IL-12 and IL-23 production in human Jurkat T cells in response to Toxoplasma gondii and LPS. IL-12 and IL-23 production was significantly increased in T cells after stimulation with T. gondii or LPS. T. gondii and LPS increased the phosphorylation of AKT, ERK1/2, p38 MAPK, and JNK1/2 in T cells from 10 min post-stimulation, and peaked at 30–60 min. Inhibition of the PI3K pathway reduced IL-12 and IL-23 production in T. gondii-infected cells, but increased in LPS-stimulated cells. IL-12 and IL-23 production was significantly reduced by ERK1/2 and p38 MAPK inhibitors in T. gondii- and LPS-stimulated cells, but not in cells treated with a JNK1/2 inhibitor. Collectively, IL-12 and IL-23 production was positively regulated by PI3K and JNK1/2 in T. gondii-infected Jurkat cells, but negatively regulated in LPS-stimulated cells. And ERK1/2 and p38 MAPK positively regulated IL-12 and IL-23 production in Jurkat T cells. These data indicate that T. gondii and LPS induced IL-12 and IL-23 production in Jurkat T cells through the regulation of the PI3K and MAPK pathways; however, the mechanism underlying the stimulation of IL-12 and IL-23 production by T. gondii in Jurkat T cells is different from that of LPS.

Small Heterodimer Partner and Innate Immune Regulation

The nuclear receptor superfamily consists of the steroid and non-steroid hormone receptors and the orphan nuclear receptors. Small heterodimer partner (SHP) is an orphan family nuclear receptor that plays an essential role in the regulation of glucose and cholesterol metabolism. Recent studies reported a previously unidentified role for SHP in the regulation of innate immunity and inflammation. The innate immune system has a critical function in the initial response against a variety of microbial and danger signals. Activation of the innate immune response results in the induction of inflammatory cytokines and chemokines to promote anti-microbial effects. An excessive or uncontrolled inflammatory response is potentially harmful to the host, and can cause tissue damage or pathological threat. Therefore, the innate immune response should be tightly regulated to enhance host defense while preventing unwanted immune pathologic responses. In this review, we discuss recent studies showing that SHP is involved in the negative regulation of toll-like receptor-induced and NLRP3 (NACHT, LRR and PYD domains-containing protein 3)-mediated inflammatory responses in innate immune cells. Understanding the function of SHP in innate immune cells will allow us to prevent or modulate acute and chronic inflammation processes in cases where dysregulated innate immune activation results in damage to normal tissues.

Involvement of PI3K/AKT and MAPK Pathways for TNF-alpha Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis

Trichomonas vaginalis induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-alpha production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-alpha production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-alpha production was significantly decreased compared to the control; however, TNF-alpha reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-alpha production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-alpha production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.

Characterization of Proinflammatory Responses and Innate Signaling Activation in Macrophages Infected with Mycobacterium scrofulaceum

Mycobacterium scrofulaceum is an environmental and slow-growing atypical mycobacterium. Emerging evidence suggests that M. scrofulaceum infection is associated with cervical lymphadenitis in children and pulmonary or systemic infections in immunocompromised adults. However, the nature of host innate immune responses to M. scrofulaceum remains unclear. In this study, we examined the innate immune responses in murine bone marrow-derived macrophages (BMDMs) infected with different M. scrofulaceum strains including ATCC type strains and two clinically isolated strains (rough and smooth types). All three strains resulted in the production of proinflammatory cytokines in BMDMs mediated through toll-like receptor-2 and the adaptor MyD88. Activation of MAPKs (extracellular signal-regulated kinase 1/2, and p38, and c-Jun N-terminal kinase) and nuclear receptor (NF)-kappaB together with intracellular reactive oxygen species generation were required for the expression of proinflammatory cytokines in BMDMs. In addition, the rough morphotypes of M. scrofulaceum clinical strains induced higher levels of proinflammatory cytokines, MAPK and NF-kappaB activation, and ROS production than other strains. When mice were infected with different M. scrofulaceum strains, those infected with the rough strain showed the greatest hepatosplenomegaly, granulomatous lesions, and immune cell infiltration in the lungs. Notably, the bacterial load was higher in mice infected with rough colonies than in mice infected with ATCC or smooth strains. Collectively, these data indicate that rough M. scrofulaceum induces higher inflammatory responses and virulence than ATCC or smooth strains.

Host immune responses to mycobacterial antigens and their implications for the development of a vaccine to control tuberculosis

Tuberculosis (TB) remains a worldwide health problem, causing around 2 million deaths per year. Despite the bacillus Calmette Guerin vaccine being available for more than 80 years, it has limited effectiveness in preventing TB, with inconsistent results in trials. This highlights the urgent need to develop an improved TB vaccine, based on a better understanding of host-pathogen interactions and immune responses during mycobacterial infection. Recent studies have revealed a potential role for autophagy, an intracellular homeostatic process, in vaccine development against TB, through enhanced immune activation. This review attempts to understand the host innate immune responses induced by a variety of protein antigens from Mycobacterium tuberculosis, and to identify future vaccine candidates against TB. We focus on recent advances in vaccine development strategies, through identification of new TB antigens using a variety of innovative tools. A new understanding of the host-pathogen relationship, and the usefulness of mycobacterial antigens as novel vaccine candidates, will contribute to the design of the next generation of vaccines, and to improving the host protective immune responses while limiting immunopathology during M. tuberculosis infection.

Autophagy and bacterial infectious diseases

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.

Toll-like Receptors and Innate Immunity

Toll-like receptors (TLRs) are the best-characterized membrane-bound receptors in innate immune cells, including macrophages and dendritic cells. Upon recognition of specific ligands originating from pathogen- and modified self-derived molecules, TLRs trigger intracellular signaling cascades that involve various adaptor proteins and enzymes, resulting in the generation of proinflammatory and antimicrobial responses through the activation of transcription factors such as nuclear factor-kappaB. TLR-dependent signaling pathways are tightly regulated during innate immune responses by a variety of negative regulators. This review focuses on the newly described regulation of TLR-dependent signaling pathways, and emphasizes the roles of TLRs in innate immunity. Efforts to modulate these regulatory pathways and signaling molecules may result in the development of new therapeutic strategies through TLR-based therapy.

Nitric Oxide Synthesis is Modulated by 1,25-Dihydroxyvitamin D3 and Interferon-gamma in Human Macrophages after Mycobacterial Infection

BACKGROUND: 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. METHODS: 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. RESULTS: Here we show that combined treatment with 1,25-dihydroxyvitamin D3 (1,25-D3) and Interferon (IFN)-gamma 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-kappaB 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-gamma 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. CONCLUSION: These data support a key role of NO in host defenses against TB and identify novel modulating factors for iNOS induction in human macrophages.

The Role of Nitric Oxide in Mycobacterial Infections

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.