Host Proinflammatory Cytokines Research Articles

Aspergillus fumigatus conidial surface-associated proteome reveals factors for fungal evasion and host immunity modulation.

Aspergillus fumigatus causes aspergillosis and relies on asexual spores (conidia) for initiating host infection. There is scarce information about A. fumigatus proteins involved in fungal evasion and host immunity modulation. Here we analysed the conidial surface proteome of A. fumigatus, two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis, as well as pathogenic Aspergillus lentulus, to identify such proteins. After identifying 62 proteins exclusively detected on the A. fumigatus conidial surface, we assessed null mutants for 42 genes encoding these proteins. Deletion of 33 of these genes altered susceptibility to macrophage, epithelial cells and cytokine production. Notably, a gene that encodes a putative glycosylasparaginase, modulating levels of the host proinflammatory cytokine IL-1β, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins are important for evasion and modulation of the immune response at the onset of fungal infection.

Monoterpene antifungal activities: evaluating geraniol, citronellal, and linalool on Candida biofilm, host inflammatory responses, and structure-activity relationships.

Introduction: Despite the rising concern with fungal resistance, a myriad of molecules has yet to be explored. Geraniol, linalool, and citronellal are monoterpenes with the same molecular formula (C10H18O), however, neither the effect of these compounds on inflammatory axis induced by Candida spp. nor the antibiofilm Structure-Activity Relationship (SAR) have been well-investigated. Herein we analyzed geraniol, linalool and citronellal antifungal activity, cytotoxicity, and distinctive antibiofilm SAR, also the influence of geraniol on Candida spp induced dysregulated inflammatory axis, and in vivo toxicity. Methods: Minimal inhibitory (MIC) and fungicidal (MFC) concentrations against Candida spp were defined, followed by antibiofilm activity (CFU-colony forming unit/mL/g of dry weight). Cytotoxic activity was assessed using human monocytes (THP-1) and oral squamous cell (TR146). Geraniol was selected for further analysis based on antifungal, antibiofilm and cytotoxic results. Geraniol was tested using a dual-chamber co-culture model with TR146 cells infected with C. albicans, and THP-1 cells, used to mimic oral epithelium upon fungal infection. Expression of Candida enzymes (phospholipase-PLB and aspartyl proteases-SAP) and host inflammatory cytokines (interleukins: IL-1β, IL-6, IL-17, IL-18, IL-10, and Tumor necrosis factor-TNF) were analyzed. Lastly, geraniol in vivo toxicity was assessed using Galleria mellonella. Results: MIC values obtained were 1.25-5mM/mL for geraniol, 25-100mM/mL for linalool, and 100-200mM/mL for citronellal. Geraniol 5 and 50mM/mL reduced yeast viability during biofilm analysis, only 500mM/mL of linalool was effective against a 72h biofilm and no biofilm activity was seen for citronellal. LD50 for TR146 and THP-1 were, respectively: geraniol 5.883 and 8.027mM/mL; linalool 1.432 and 1.709mM/mL; and citronellal 0.3006 and 0.1825mM/mL. Geraniol was able to downregulate expression of fungal enzymes and host pro-inflammatory cytokines IL-1β, IL-6, and IL-18. Finally, safety in vivo parameters were observed up to 20mM/Kg. Discussion: Despite chemical similarities, geraniol presented better antifungal, antibiofilm activity, and lower cytotoxicity when compared to the other monoterpenes. It also showed low in vivo toxicity and capacity to downregulate the expression of fungal enzymes and host pro-inflammatory cytokines. Thus, it can be highlighted as a viable option for oral candidiasis treatment.

Latest Insights on the Diagnostic Approaches and Treatment Strategies of COVID-19.

BackgroundCOVID-19 has emerged as the most serious pandemic in the 21st century to date. COVID-19 patients may develop various disease symptoms that hinder the accurate clinical diagnosis.SummaryRoutine diagnosis of COVID-19 requires complementary investigations, including computed tomography, immunological assays, and molecular assays like real-time RT-PCR, loop-mediated isothermal amplification, metagenomic next-generation sequencing, and clusters of regularly interspaced short palindromic repeats-based assays. Clinically approved antiviral drugs available for the COVID-19 treatment are very limited. The most common measurements that enhance health condition and patients' viability are conservation fluid management, oxygen therapy, and antibiotics. Several therapeutic options have been developed or repurposed to prevent virus replication and/or modulate the immune response against virus infection. These options include various drugs that affect virus entry and membrane fusion, inhibit polymerase and protease activity, suppress the host pro-inflammatory cytokines, and utilize cell therapy approaches.Key MessagesIn this review, we aimed to provide an up-to-date discussion on the current diagnostic options and therapeutic strategies used to control and manage COVID-19 in clinical and point-of-care settings.

Peptides derived from the SARS-CoV-2 receptor binding motif bind to ACE2 but do not block ACE2-mediated host cell entry or pro-inflammatory cytokine induction.

SARS-CoV-2 viral attachment and entry into host cells is mediated by a direct interaction between viral spike glycoproteins and membrane bound angiotensin-converting enzyme 2 (ACE2). The receptor binding motif (RBM), located within the S1 subunit of the spike protein, incorporates the majority of known ACE2 contact residues responsible for high affinity binding and associated virulence. Observation of existing crystal structures of the SARS-CoV-2 receptor binding domain (SRBD)-ACE2 interface, combined with peptide array screening, allowed us to define a series of linear native RBM-derived peptides that were selected as potential antiviral decoy sequences with the aim of directly binding ACE2 and attenuating viral cell entry. RBM1 (16mer): S443KVGGNYNYLYRLFRK458, RBM2A (25mer): E484GFNCYFPLQSYGFQPTNGVGYQPY508, RBM2B (20mer): F456NCYFPLQSYGFQPTNGVGY505 and RBM2A-Sc (25mer): NYGLQGSPFGYQETPYPFCNFVQYG. Data from fluorescence polarisation experiments suggested direct binding between RBM peptides and ACE2, with binding affinities ranging from the high nM to low μM range (Kd = 0.207-1.206 μM). However, the RBM peptides demonstrated only modest effects in preventing SRBD internalisation and showed no antiviral activity in a spike protein trimer neutralisation assay. The RBM peptides also failed to suppress S1-protein mediated inflammation in an endogenously expressing ACE2 human cell line. We conclude that linear native RBM-derived peptides are unable to outcompete viral spike protein for binding to ACE2 and therefore represent a suboptimal approach to inhibiting SARS-CoV-2 viral cell entry. These findings reinforce the notion that larger biologics (such as soluble ACE2, 'miniproteins', nanobodies and antibodies) are likely better suited as SARS-CoV-2 cell-entry inhibitors than short-sequence linear peptides.

Interception of host fatty acid metabolism by mycobacteria under hypoxia to suppress anti-TB immunity

Pathogenic mycobacteria induce the formation of hypoxic granulomas during latent tuberculosis (TB) infection, in which the immune system contains, but fails to eliminate the mycobacteria. Fatty acid metabolism-related genes are relatively overrepresented in the mycobacterial genome and mycobacteria favor host-derived fatty acids as nutrient sources. However, whether and how mycobacteria modulate host fatty acid metabolism to drive granuloma progression remains unknown. Here, we report that mycobacteria under hypoxia markedly secrete the protein Rv0859/MMAR_4677 (Fatty-acid degradation A, FadA), which is also enriched in tuberculous granulomas. FadA acts as an acetyltransferase that converts host acetyl-CoA to acetoacetyl-CoA. The reduced acetyl-CoA level suppresses H3K9Ac-mediated expression of the host proinflammatory cytokine Il6, thus promoting granuloma progression. Moreover, supplementation of acetate increases the level of acetyl-CoA and inhibits the formation of granulomas. Our findings suggest an unexpected mechanism of a hypoxia-induced mycobacterial protein suppressing host immunity via modulation of host fatty acid metabolism and raise the possibility of a novel therapeutic strategy for TB infection.

Melanin of Sporothrix globosa affects the function of THP-1 macrophages and modulates the expression of TLR2 and TLR4

BackgroundMelanin is an important virulence factor for Sporothrix globosa, the causative agent of sporotrichosis, a subcutaneous mycosis that occurs worldwide. Although previous research suggests that melanin is involved in the pathogenesis of sporotrichosis, little is known about its influence on the macrophages that represent the frontline components of innate immunity. ObjectivesTo evaluate the effects of melanin on phagocytic activity and the expression of Toll-like receptor (TLR)2 and TLR4 during S. globosa infection of macrophages in vitro. MethodsTo compare phagocytic activity and survival rates, THP-1 macrophages and primary mouse peritoneal macrophages were co-cultured with a wild-type S. globosa strain (Mel+), an albino mutant strain (Mel˗), a tricyclazole-treated Mel + strain (TCZ-Mel+), or melanin ghosts extracted from S. globosa conidia. Reactive oxygen species (ROS), nitric oxide (NO) generation, tumor necrosis factor (TNF)-α and interleukin (IL)-6 were assayed in THP-1 cells infected with S. globosa conidia. Quantitative PCR and western blotting were used to observe the effect of melanin on TLR2 and TLR4 expression. Knockdown of TLR2/4 expression with small interfering RNA was performed to further verify the role of these receptors during infection. ResultsMacrophages infected with Mel + conidia showed a lower phagocytosis index and a higher survival rate than TCZ-Mel+ and Mel˗ in vitro. After incubation with S. globosa, the release of ROS, NO, TNF-α and IL-6 by THP-1 were decreased in the presence of melanin. Increased mRNA and protein expression of TLR2 and TLR4 occurred upon S. globosa infection in THP-1, whereas the presence of melanin suppressed TLR2 and TLR4. Moreover, TLR2 or TLR4 knockdown showed a trend toward reducing the pernicious effect of S. globosa conidia on THP-1 cells in vitro. ConclusionsCollectively, our results indicated that melanin inhibits the phagocytosis of S. globosa and guards against macrophage attack by providing protection from oxygen- and nitrogen-derived radicals, as well as suppressing the host pro-inflammatory cytokine response (TNF-α and IL-6). Melanin was also involved in modulating TLR2 and TLR4 receptor expression, weakening the killing efficiency of S. globosa.

Giardia duodenalis Induces Proinflammatory Cytokine Production in Mouse Macrophages via TLR9-Mediated p38 and ERK Signaling Pathways.

Giardia duodenalis, also known as Giardia lamblia or Giardia intestinalis, is an important opportunistic, pathogenic, zoonotic, protozoan parasite that infects the small intestines of humans and animals, causing giardiasis. Several studies have demonstrated that innate immunity-associated Toll-like receptors (TLRs) are critical for the elimination of G. duodenalis; however, whether TLR9 has a role in innate immune responses against Giardia infection remains unknown. In the present study, various methods, including reverse transcriptase–quantitative polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, immunofluorescence, inhibitor assays, and small-interfering RNA interference, were utilized to probe the role of TLR9 in mouse macrophage-mediated defenses against G. lamblia virus (GLV)–free or GLV-containing Giardia trophozoites. The results revealed that in G. duodenalis–stimulated mouse macrophages, the secretion of proinflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-12 p40, was enhanced, concomitant with the significant activation of TLR9, whereas silencing TLR9 attenuated the host inflammatory response. Notably, the presence of GLV exacerbated the secretion of host proinflammatory cytokines. Moreover, G. duodenalis stimulation activated multiple signaling pathways, including the nuclear factor κB p65 (NF-κB p65), p38, ERK, and AKT pathways, the latter three in a TLR9-dependent manner. Additionally, inhibiting the p38 or ERK pathway downregulated the G. duodenalis–induced inflammatory response, whereas AKT inhibition aggravated this process. Taken together, these results indicated that G. duodenalis may induce the secretion of proinflammatory cytokines by activating the p38 and ERK signaling pathways in a TLR9-dependent manner in mouse macrophages. Our in vitro findings on the mechanism underlying the TLR9-mediated host inflammatory response may help establish the foundation for an in-depth investigation of the role of TLR9 in the pathogenicity of G. duodenalis.

Influence of Hypoxia on the Epithelial-Pathogen Interactions in the Lung: Implications for Respiratory Disease.

Under normal physiological conditions, the lung remains an oxygen rich environment. However, prominent regions of hypoxia are a common feature of infected and inflamed tissues and many chronic inflammatory respiratory diseases are associated with mucosal and systemic hypoxia. The airway epithelium represents a key interface with the external environment and is the first line of defense against potentially harmful agents including respiratory pathogens. The protective arsenal of the airway epithelium is provided in the form of physical barriers, and the production of an array of antimicrobial host defense molecules, proinflammatory cytokines and chemokines, in response to activation by receptors. Dysregulation of the airway epithelial innate immune response is associated with a compromised immunity and chronic inflammation of the lung. An increasing body of evidence indicates a distinct role for hypoxia in the dysfunction of the airway epithelium and in the responses of both innate immunity and of respiratory pathogens. Here we review the current evidence around the role of tissue hypoxia in modulating the host-pathogen interaction at the airway epithelium. Furthermore, we highlight the work needed to delineate the role of tissue hypoxia in the pathophysiology of chronic inflammatory lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease in addition to novel respiratory diseases such as COVID-19. Elucidating the molecular mechanisms underlying the epithelial-pathogen interactions in the setting of hypoxia will enable better understanding of persistent infections and complex disease processes in chronic inflammatory lung diseases and may aid the identification of novel therapeutic targets and strategies.

Deletion of the lon gene augments expression of Salmonella Pathogenicity Island (SPI)-1 and metal ion uptake genes leading to the accumulation of bactericidal hydroxyl radicals and host pro-inflammatory cytokine-mediated rapid intracellular clearance

ABSTRACT In the present study, we characterized the involvement of Lon protease in bacterial virulence and intracellular survival in Salmonella under abiotic stress conditions resembling the conditions of a natural infection. Wild type (JOL401) and the lon mutant (JOL909) Salmonella Typhimurium were exposed to low temperature, pH, osmotic, and oxidative stress conditions and changes in gene expression profiles related to virulence and metal ion uptake were investigated. Expression of candidate genes invF and hilC of Salmonella Pathogenicity Island (SPI)-1 and sifA and sseJ of SPI-2 revealed that Lon protease controls SPI-1 genes and not SPI-2 genes under all stress conditions tested. The lon mutant exhibited increased accumulation of hydroxyl (OH·) ions that lead to cell damage due to oxidative stress. This oxidative damage can also be linked to an unregulated influx of iron due to the upregulation of ion channel genes such as fepA in the lon mutant. The deletion of lon from the Salmonella genome causes oxidative damage and increased expression of virulence genes. It also prompts the secretion of host pro-inflammatory cytokines leading to early clearance of the bacteria from host cells. We conclude that poor bacterial recovery from mice infected with the lon mutant is a result of disrupted bacterial intracellular equilibrium and rapid activation of cytokine expression leading to bacterial lysis.

Mycobacterium tuberculosis Rv3717 enhances the survival of Mycolicibacterium smegmatis by inhibiting host innate immune and caspase-dependent apoptosis

Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) infection remains a serious public threat despite decades of creative endeavors. There are few reports on the roles of M. tuberculosis enzymes involved in cell envelope biosynthesis in pathogen survival and persistence. M. tuberculosis Rv3717 encodes N-acetylmuramoyl-l-alanine amidase, a cell-wall hydrolase that hydrolyzes the bond between N-acetylmuramic acid and l-alanine in cell-wall peptidoglycan. In this paper, we demonstrated the Rv3717 promoted the survival of Mycolicibacterium smegmatis(M. smegmatis) within macrophages. More importantly, we demonstrated that this effect is because MS_Rv3717 reduces the release of host pro-inflammatory cytokines such as IL-1β, IL-6, IL-12 p40, TNF-α, and increased transcription of anti-inflammatory cytokine IL-10. At the same time, MS_Rv3717 inhibits apoptosis by inhibiting the activation of Caspase-3/9, reducing the host's elimination of M. smegmatis. Finally, from a bacterial perspective, we found Rv3717 decreased the survival of M. smegmatis under stresses such as SDS and low pH. This is the first report of the involvement of Mycobacterium cell envelope biosynthetic enzyme in host-pathogen interaction.

Erythrocyte-Coated Nanoparticles Block Cytotoxic Effects of Group B Streptococcus β-Hemolysin/Cytolysin.

Group B Streptococcus (GBS) emerged as a leading cause of invasive infectious disease in neonates in the 1970s, but has recently been identified as an escalating public health threat in non-pregnant adults, particularly those of advanced aged or underlying medical conditions. GBS infection can rapidly develop into life-threatening disease despite prompt administration of effective antibiotics and initiation of state-of-the-art intensive care protocols and technologies due to deleterious bacterial virulence factors, such as the GBS pore-forming toxin β-hemolysin/cytolysin (β-H/C). β-H/C is known to have noxious effects on a wide range of host cells and tissues, including lung epithelial cell injury, blood brain barrier weakening, and immune cell apoptosis. Neonatal and adult survivors of GBS infection are at a high risk for substantial long-term health issues and neurologic disabilities due to perturbations in organ systems caused by bacterial- and host- mediated inflammatory stressors. Previously engineered anti-virulence inhibitors, such as monoclonal antibodies and small molecular inhibitors, generally require customized design for each different pathogenic toxin and do not target deleterious host pro-inflammatory responses that may cause organ injury, septic shock, or death. By simply wrapping donor red blood cells (RBCs) around polymeric cores, we have created biomimetic “nanosponges.” Because nanoparticles retain the same repertoire of cell membrane receptors as their host cell, they offer non-specific and all-purpose toxin decoy strategies with a broad ability to sequester and neutralize various bacterial toxins and host pro-inflammatory chemokines and cytokines to attenuate the course of infectious disease. This proof-of-concept study successfully demonstrated that intervention with nanosponges reduced the hemolytic activity of live GBS and stabilized β-H/C in a dose-dependent manner. Nanosponge treatment also decreased lung epithelial and macrophage cell death following exposure to live GBS bacteria and stabilized β-H/C, improved neutrophil killing of GBS, and diminished GBS-induced macrophage IL-1β production. Our results, therefore, suggest biomimetic nanosponges provide a titratable detoxification therapy that may provide a first-in-class treatment option for GBS infection by sequestering and inhibiting β-H/C activity.

Neisseria meningitidis-mediates proinflammatory responses in PMN: Crosstalk TLR-2/c-JunN-Terminal

Neisseria meningitidis is a Gram-negative bacterium emerging the leading cause of bacterial meningitis in children and young adult wide world. The host innate immune response against meningitis is largely unknown. In this study, we show that N.meningitidis robustly activates mRNA and protein expression of tumor necrosis factor (TNF-α) and interleukin (IL-6) in murine bone marrow-derived PMN. Toll-like receptor (TLR-2) and myeloid differentiation primary response gene 88 (MyD88), N.meningitidis also activates the mitogen-activated protein kinase (MAPKs; c-Jun N-terminal kinase (JNK), ERK1/2 and p38 MAPK) pathway. N.meningitidis-induced TNF-α and IL-6 production was dependent on JNK activation. The intracellular reactive oxygen species (ROS), NADPH oxidase-2, and nuclear factor-κB are required for N.meningitides-induced proinflammatory cytokine generation in PMN. Together, we have demonstrated that N.meningitidis-induced activation of host proinflammatory cytokines is mediated through TLR2-dependent JNK signaling pathways.

Peripheral Francisella Tularensis Infection Results In Neural Invasion and Pathologic Inflammation

Abstract Francisella tularensis infection triggers an overactive inflammatory response by stimulating production of host pro-inflammatory cytokines. Human tularemia, caused by F. tularensis, is a zoonotic disease transmitted by aerosol particles, direct contact, and via arthropod vectors, particularly ticks. With the known peripheral invasion of F. tularensis, and previously published clinical data indicating the presence of our bacterium in cerebral spinal fluid, many questions arise, which include the questions as to which specific neuronal cells are infected. In the periphery, macrophages provide innate immune defense against foreign material, including bacteria. Microglial cells are resident macrophages of the brain and spinal cord and previously have been reported to rapidly respond to pathological changes in the central nervous system, serving a similar function as macrophages. Their rapid activation is an important factor in guarding the neural parenchyma against infectious diseases, inflammation, and neurodegeneration while maintaining and facilitating the return to tissue homeostasis. Therefore, we hypothesized that, having invaded the brain by an unknown mechanism, F. tularensis may be capable of eliciting an immune response following infection of microglial cells. Since macrophage infection leads to elicitation of inflammation, infection of microglia similarly stimulate inflammation leading to neural pathology. Mice were inoculated on the rear flank with fluorescently labeled bacteria. Using fluorescent confocal microscopy, labeled F. tularensis was localized in the brains of infected mice. We analyzed brain homogenates for production of inflammatory cytokines at these sites through Milliplex and qPCR.

Peripheral Francisella Tularensis Infection Results In Neural Invasion and Pathologic Inflammation

Abstract Francisella tularensis infection triggers an overactive inflammatory response by stimulating production of host pro-inflammatory cytokines. Human tularemia, caused by F. tularensis, is a zoonotic disease transmitted by aerosol particles, direct contact, and via arthropod vectors, particularly ticks. With the known peripheral invasion of F. tularensis, and previously published clinical data indicating the presence of our bacterium in cerebral spinal fluid, many questions arise, which include the questions as to which specific neuronal cells are infected. In the periphery, macrophages provide innate immune defense against foreign material, including bacteria. Microglial cells are resident macrophages of the brain and spinal cord and previously have been reported to rapidly respond to pathological changes in the central nervous system, serving a similar function as macrophages. Their rapid activation is an important factor in guarding the neural parenchyma against infectious diseases, inflammation, and neurodegeneration while maintaining and facilitating the return to tissue homeostasis. Therefore, we hypothesized that, having invaded the brain by an unknown mechanism, F. tularensis may be capable of eliciting an immune response following infection of microglial cells. Since macrophage infection leads to elicitation of inflammation, infection of microglia similarly stimulate inflammation leading to neural pathology. Mice were inoculated on the rear flank with fluorescently labeled bacteria. Using fluorescent confocal microscopy, labeled F. tularensis was localized in the brains of infected mice. We analyzed brain homogenates for production of inflammatory cytokines at these sites through Milliplex and qPCR.

TLR7/8 agonist induces a post-entry SAMHD1-independent block to HIV-1 infection of monocytes.

BackgroundMonocytes, the primary myeloid cell-type in peripheral blood, are resistant to HIV-1 infection as a result of the lentiviral restriction factor SAMHD1. Toll-like receptors recognize microbial pathogen components, inducing the expression of antiviral host proteins and proinflammatory cytokines. TLR agonists that mimic microbial ligands have been found to have activity against HIV-1 in macrophages. The induction of restriction factors in monocytes by TLR agonist activation has not been well studied. To analyze restriction factor induction by TLR activation in monocytes, we used the imidazoquinoline TLR7/8 agonist R848 and infected with HIV-1 reporter virus that contained packaged viral accessory protein Vpx, which allows the virus to escape SAMHD1-mediated restriction. ResultsR848 prevented the replication of Vpx-containing HIV-1 and HIV-2 in peripheral blood mononuclear cells and monocytes. The block was post-entry but prior to reverse transcription of the viral genomic RNA. The restriction was associated with destabilization of the genomic RNA molecules of the in-coming virus particle. R848 treatment of activated T cells did not protect them from infection but treated monocytes produced high levels of proinflammatory cytokines, including type-I IFN that protected bystander activated T cells from infection.ConclusionThe activation of TLR7/8 induces two independent restrictions to HIV-1 replication in monocytes: a cell-intrinsic block that acts post-entry to prevent reverse transcription; and a cell-extrinsic block, in which monocytes produce high levels of proinflammatory cytokines (primarily type-I IFN) that protects bystander monocytes and T lymphocytes. The cell-intrinsic block may result from the induction of a novel restriction factor, which can be termed Lv5 and acts by destabilizing the in-coming viral genomic RNA, either by the induction of a host ribonuclease or by disrupting the viral capsid. TLR agonists are being developed for therapeutic use to diminish the size of the latent provirus reservoir in HIV-1 infected individuals. Such drugs may both induce latent provirus expression and restrict virus replication during treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0316-3) contains supplementary material, which is available to authorized users.

Host Intracellular Signaling Events and Pro-inflammatory Cytokine Production in African Trypanosomiasis.

Pathogens, such as bacteria, viruses, and parasites, possess specific molecules or proteins that are recognized by several host innate immune receptors, leading to the activation of several intracellular signaling molecules and pathways. The magnitude and quality of these events significantly affect the outcome of infection. African trypanosomes, including Trypanosoma congolense, are capable of manipulating the host immune response, including the activity of macrophages, which are the key immune cells that contribute to the immunopathogenesis of African trypanosomiasis. Although it is known that immune hyperactivation and excessive pro-inflammatory cytokine production are the hallmarks of African trypanosomiasis, the mechanisms through which these events are triggered are poorly defined. However, it is known that macrophages may play a significant role in these processes, because phagocytosis of trypanosomes by macrophages initiates intracellular signal transduction cascades that lead to the release of pro-inflammatory cytokines and alteration in cell function. This review highlights recent progress in our understanding of the innate immune receptors, signaling pathways, and transcription factors involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. It will reveal the existence of complex signaling events through which the parasite modulates the host immune response, thus identifying novel targets that could aid in designing strategies to effectively control the disease.

Peripheral Francisella tularensis infection results in neural invasion and pathologic inflammation.

Abstract Francisella tularensis infection triggers an overactive inflammatory response by stimulating production of host pro-inflammatory cytokines. Human tularemia, caused by F. tularensis, is a zoonotic disease transmitted by aerosol particles, direct contact, and via arthropod vectors, particularly ticks. Curiously, during peripheral inoculation in a mouse model of tularemia, F. tularensis was unexpectedly found in brain homogenates, though there is no prior evidence suggesting neural tropism. In the periphery, macrophages provide innate immune defense against foreign material, including bacteria. Microglial cells are resident macrophages of the brain and spinal cord and previously have been reported to rapidly respond to pathological changes in the central nervous system, serving a similar function as macrophages. Their rapid activation is an important factor in guarding the neural parenchyma against infectious diseases, inflammation, and neurodegeneration while maintaining and facilitating the return to tissue homeostasis. Therefore, we hypothesized that, having invaded the brain by an unknown mechanism, F. tularensis may be capable of eliciting an immune response following infection of microglial cells. Since macrophage infection leads to elicitation of inflammation, infection of microglia similarly stimulate inflammation leading to neural pathology. Mice were inoculated on the rear flank with fluorescently labeled bacteria. Using fluorescent confocal microscopy, labeled F. tularensis was localized in the brains of infected mice. We analyzed brain homogenates for production of inflammatory cytokines at these sites through Milliplex and qPCR.

A coagulase-negative and non-haemolytic strain of Staphylococcus aureus for investigating the roles of SrtA in a murine model of bloodstream infection.

Sortase A (SrtA) is a cysteine transpeptidase and virulence factor from Staphylococcus aureus (S. aureus) that catalyses the attachment and display of surface proteins on the cell wall, thereby mediating bacterial adhesion to host tissues, host-cell entry and evasion of the immune response. As a result, SrtA has become an important target in the development of therapies for S. aureus infections. In this study, we used the new reference strain S. aureus Newman D2C to investigate the role of SrtA in a murine model of bloodstream infection, when the impact of coagulase and haemolysin is excluded. The results suggested that deletion of SrtA reduced the bacterial burden on the heart, liver and kidneys by blunting the host proinflammatory cytokine response at an early point in infection. Kidneys, but not heart or liver, formed abscesses on the sixth day following non-lethal infection, and this effect was diminished by SrtA mutation. These findings indicate that SrtA is a determining virulence factor in lethality and formation of renal abscesses in mice followed by S. aureus bloodstream infection. We have thus established a convenient in vitro and mouse model for developing SrtA-targeted therapeutic strategies.

Mycobacterium tuberculosis Rv3402c enhances mycobacterial survival within macrophages and modulates the host pro-inflammatory cytokines production via NF-kappa B/ERK/p38 signaling.

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis, a process which depends on an array of virulence factors to colonize and replicate within the host. The M. tuberculosis iron regulated open reading frame (ORF) rv3402c, encoding a conserved hypothetical protein, was shown to be up-regulated upon infection in both human and mice macrophages. To explore the function of this ORF, we heterologously expressed the rv3402c gene in the non-pathogenic fast-growing Mycobacterium smegmatis strain, and demonstrated that Rv3402c, a cell envelope-associated protein, was able to enhance the intracellular survival of recombinant M. smegmatis. Enhanced growth was not found to be the result of an increased resistance to intracellular stresses, as growth of the Rv3402c expressing strain was unaffected by iron depletion, H2O2 exposure, or acidic conditions. Colonization of macrophages by M. smegmatis expressing Rv3402c was associated with substantial cell death and significantly greater amount of TNF-α and IL-1β compared with controls. Rv3402c-induced TNF-α and IL-1β production was found to be mediated by NF-κB, ERK and p38 pathway in macrophages. In summary, our study suggests that Rv3402c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophage, promote host cell death and enhance the persistence of mycobacterium within host cells.

Mycobacterium massiliense Induces Inflammatory Responses in Macrophages Through Toll-Like Receptor 2 and c-Jun N-Terminal Kinase

Mycobacterium massiliense (Mmass) is an emerging, rapidly growing mycobacterium (RGM) that belongs to the M. abscessus (Mabc) group, albeit clearly differentiated from Mabc. Compared with M. tuberculosis, a well-characterized human pathogen, the host innate immune response against Mmass infection is largely unknown. In this study, we show that Mmass robustly activates mRNA and protein expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in murine bone marrow-derived macrophages (BMDMs). Toll-like receptor (TLR)-2 and myeloid differentiation primary response gene 88 (MyD88), but neither TLR4 nor Dectin-1, are involved in Mmass-induced TNF-α or IL-6 production in BMDMs. Mmass infection also activates the mitogen-activated protein kinase (MAPKs; c-Jun N-terminal kinase (JNK), ERK1/2 and p38 MAPK) pathway. Mmass-induced TNF-α and IL-6 production was dependent on JNK activation, while they were unaffected by either the ERK1/2 or p38 pathway in BMDMs. Additionally, intracellular reactive oxygen species (ROS), NADPH oxidase-2, and nuclear factor-κB are required for Mmass-induced proinflammatory cytokine generation in macrophages. Furthermore, the S morphotype of Mmass showed lower overall induction of pro-inflammatory (TNF-α, IL-6, and IL-1β) and anti-inflammatory (IL-10) cytokines than the R morphotype, suggesting fewer immunogenic characteristics for this clinical strain. Together, these results suggest that Mmass-induced activation of host proinflammatory cytokines is mediated through TLR2-dependent JNK and ROS signaling pathways.