Interferon-γ Is a Crucial Activator of Early Host Immune Defense against Mycobacterium ulcerans Infection in Mice

Buruli ulcer (BU), caused by infection with Mycobacterium ulcerans, is a chronic necrotizing human skin disease associated with the production of the cytotoxic macrolide exotoxin mycolactone. Despite extensive research, the type of immune responses elicited against this pathogen and the effector functions conferring protection against BU are not yet fully understood. While histopathological analyses of advanced BU lesions have demonstrated a mainly extracellular localization of the toxin producing acid fast bacilli, there is growing evidence for an early intra-macrophage growth phase of M. ulcerans. This has led us to investigate whether interferon-γ might play an important role in containing M. ulcerans infections. In an experimental Buruli ulcer mouse model we found that interferon-γ is indeed a critical regulator of early host immune defense against M. ulcerans infections. Interferon-γ knockout mice displayed a faster progression of the infection compared to wild-type mice. This accelerated progression was reflected in faster and more extensive tissue necrosis and oedema formation, as well as in a significantly higher bacterial burden after five weeks of infection, indicating that mice lacking interferon-γ have a reduced capacity to kill intracellular bacilli during the early intra-macrophage growth phase of M. ulcerans. This data demonstrates a prominent role of interferon-γ in early defense against M. ulcerans infection and supports the view that concepts for vaccine development against tuberculosis may also be valid for BU.     

The Role of Peroxisome Proliferator-Activated Receptor γ in Immune Responses to Enteroaggregative Escherichia coli Infection

Background

Enteroaggregative Escherichia coli (EAEC) is recognized as an emerging cause of persistent diarrhea and enteric disease worldwide. Mucosal immunity towards EAEC infections is incompletely understood due in part to the lack of appropriate animal models. This study presents a new mouse model and investigates the role of peroxisome proliferator-activated receptor gamma (PPARγ) in the modulation of host responses to EAEC in nourished and malnourished mice.

Methods/Principal Findings

Wild-type and T cell-specific PPARγ null C57BL/6 mice were fed protein-deficient diets at weaning and challenged with 5×10⁹cfu EAEC strain JM221 to measure colonic gene expression and immune responses to EAEC. Antigen-specific responses to E. coli antigens were measured in nourished and malnourished mice following infection and demonstrated the immunosuppressive effects of malnutrition at the cellular level. At the molecular level, both pharmacological blockade and deletion of PPARγ in T cells resulted in upregulation of TGF-β, IL-6, IL-17 and anti-microbial peptides, enhanced Th17 responses, fewer colonic lesions, faster clearance of EAEC, and improved recovery. The beneficial effects of PPARγ blockade on weight loss and EAEC clearance were abrogated by neutralizing IL-17 in vivo.

Conclusions

Our studies provide in vivo evidence supporting the beneficial role of mucosal innate and effector T cell responses on EAEC burden and suggest pharmacological blockade of PPARγ as a novel therapeutic intervention for EAEC infection.     

TLR–Dependent Control of Francisella tularensis Infection and Host Inflammatory Responses

Background

Francisella tularensis is the causative agent of tularemia and is classified as a Category A select agent. Recent studies have implicated TLR2 as a critical element in the host protective response to F. tularensis infection, but questions remain about whether TLR2 signaling dominates the response in all circumstances and with all species of Francisella and whether F. tularensis PAMPs are predominantly recognized by TLR2/TLR1 or TLR2/TLR6. To address these questions, we have explored the role of Toll-like receptors (TLRs) in the host response to infections with F. tularensis Live Vaccine Strain (LVS) and F. tularensis subspecies (subsp.) novicida in vivo.

Methodology/Principal Findings

C57BL/6 (B6) control mice and TLR– or MyD88-deficient mice were infected intranasally (i.n.) or intradermally (i.d.) with F. tularensis LVS or with F. tularensis subsp. novicida. B6 mice survived >21 days following infection with LVS by both routes and survival of TLR1^−/−, TLR4^−/−, and TLR6^−/− mice infected i.n. with LVS was equivalent to controls. Survival of TLR2^−/− and MyD88^−/− mice, however, was significantly reduced compared to B6 mice, regardless of the route of infection or the subspecies of F. tularensis. TLR2^−/− and MyD88^−/− mice also showed increased bacterial burdens in lungs, liver, and spleen compared to controls following i.n. infection. Primary macrophages from MyD88^−/− and TLR2^−/− mice were significantly impaired in the ability to secrete TNF and other pro-inflammatory cytokines upon ex vivo infection with LVS. TNF expression was also impaired in vivo as demonstrated by analysis of bronchoalveolar lavage fluid and by in situ immunofluorescent staining.

Conclusions/Significance

We conclude from these studies that TLR2 and MyD88, but not TLR4, play critical roles in the innate immune response to F. tularensis infection regardless of the route of infection or the subspecies. Moreover, signaling through TLR2 does not depend exclusively on TLR1 or TLR6 during F. tularensis LVS infection.     

Impact of Xenogeneic Silencing on Phage–Host Interactions

Phages, viruses that prey on bacteria, are the most abundant and diverse inhabitants of the Earth. Temperate bacteriophages can integrate into the host genome and, as so-called prophages, maintain a long-term association with their host. The close relationship between host and virus has significantly shaped microbial evolution and phage elements may benefit their host by providing new functions. Nevertheless, the strong activity of phage promoters and potentially toxic gene products may impose a severe fitness burden and must be tightly controlled. In this context, xenogeneic silencing (XS) proteins, which can recognize foreign DNA elements, play an important role in the acquisition of novel genetic information and facilitate the evolution of regulatory networks. Currently known XS proteins fall into four classes (H-NS, MvaT, Rok and Lsr2) and have been shown to follow a similar mode of action by binding to AT-rich DNA and forming an oligomeric nucleoprotein complex that silences gene expression. In this review, we focus on the role of XS proteins in phage–host interactions by highlighting the important function of XS proteins in maintaining the lysogenic state and by providing examples of how phages fight back by encoding inhibitory proteins that disrupt XS functions in the host. Sequence analysis of available phage genomes revealed the presence of genes encoding Lsr2-type proteins in the genomes of phages infecting Actinobacteria. These data provide an interesting perspective for future studies to elucidate the impact of phage-encoded XS homologs on the phage life cycle and phage–host interactions.     

In?Vivo Nonlinear Optical Imaging of Immune Responses: Tissue Injury and Infection

In this study, we demonstrate a noninvasive imaging approach based on multimodal nonlinear optical microscopy to in vivo image the responses of immune cells (neutrophils) to the tissue injury and bacterial infection in a zebrafish model. Specifically, the second harmonic generation from myosin thick filaments in sarcomere enabled a clear visualization of the muscle injury and infection. Two-photon excited fluorescence was used to track the behavior of the neutrophils that were transgenically labeled by red fluorescent protein. The corresponding reduced nicotinamide adenine dinucleotide (NADH) two-photon excited fluorescence images revealed a detailed morphological transformation process of individual neutrophils during muscle tissue injury and bacterial infection. The analysis of time-resolved NADH signals from the neutrophils provided important biological insights of the cellular energy metabolism during the immune responses. We found a significant increase of free/protein-bound NADH ratios in activated neutrophils in bacterial-infected tissue. In this study, we also discovered that, under 720 nm excitation, two wild-type strains (DH5α and BL21) of bacteria Escherichia coli emitted distinct endogenous fluorescence of double-peak at ∼450 and ∼520 nm, respectively. We demonstrated that the double-peak fluorescence signal could be used to differentiate the E. coli from surrounding tissues of dominant NADH signals, and to achieve label-free tracking of E. coli bacteria in vivo.     

Widespread Shortening of 3’ Untranslated Regions and Increased Exon Inclusion Are Evolutionarily Conserved Features of Innate Immune Responses to Infection

The contribution of pre-mRNA processing mechanisms to the regulation of immune responses remains poorly studied despite emerging examples of their role as regulators of immune defenses. We sought to investigate the role of mRNA processing in the cellular responses of human macrophages to live bacterial infections. Here, we used mRNA sequencing to quantify gene expression and isoform abundances in primary macrophages from 60 individuals, before and after infection with Listeria monocytogenes and Salmonella typhimurium. In response to both bacteria we identified thousands of genes that significantly change isoform usage in response to infection, characterized by an overall increase in isoform diversity after infection. In response to both bacteria, we found global shifts towards (i) the inclusion of cassette exons and (ii) shorter 3’ UTRs, with near-universal shifts towards usage of more upstream polyadenylation sites. Using complementary data collected in non-human primates, we show that these features are evolutionarily conserved among primates. Following infection, we identify candidate RNA processing factors whose expression is associated with individual-specific variation in isoform abundance. Finally, by profiling microRNA levels, we show that 3’ UTRs with reduced abundance after infection are significantly enriched for target sites for particular miRNAs. These results suggest that the pervasive usage of shorter 3’ UTRs is a mechanism for particular genes to evade repression by immune-activated miRNAs. Collectively, our results suggest that dynamic changes in RNA processing may play key roles in the regulation of innate immune responses.     

Behavioral and Immune Responses to Infection Require G��q- RhoA Signaling in C. elegans

Following pathogen infection the hosts'' nervous and immune systems react with coordinated responses to the danger. A key question is how the neuronal and immune responses to pathogens are coordinated, are there common signaling pathways used by both responses? Using C. elegans we show that infection by pathogenic strains of M. nematophilum, but not exposure to avirulent strains, triggers behavioral and immune responses both of which require a conserved Gαq-RhoGEF Trio-Rho signaling pathway. Upon infection signaling by the Gαq pathway within cholinergic motorneurons is necessary and sufficient to increase release of the neurotransmitter acetylcholine and increase locomotion rates and these behavioral changes result in C. elegans leaving lawns of M. nematophilum. In the immune response to infection signaling by the Gαq pathway within rectal epithelial cells is necessary and sufficient to cause changes in cell morphology resulting in tail swelling that limits the infection. These Gαq mediated behavioral and immune responses to infection are separate, act in a cell autonomous fashion and activation of this pathway in the appropriate cells can trigger these responses in the absence of infection. Within the rectal epithelium the Gαq signaling pathway cooperates with a Ras signaling pathway to activate a Raf-ERK-MAPK pathway to trigger the cell morphology changes, whereas in motorneurons Gαq signaling triggers behavioral responses independent of Ras signaling. Thus, a conserved Gαq pathway cooperates with cell specific factors in the nervous and immune systems to produce appropriate responses to pathogen. Thus, our data suggests that ligands for Gq coupled receptors are likely to be part of the signals generated in response to M. nematophilum infection.     

The Timing of IFNβ Production Affects Early Innate Responses to Listeria monocytogenes and Determines the Overall Outcome of Lethal Infection

Dendritic cells (DCs) and natural killer (NK) cells are essential components of the innate immunity and play a crucial role in the first phase of host defense against infections and tumors. Listeria monocytogenes (Lm) is an intracellular pathogen that colonizes the cytosol of eukaryotic cells. Recent findings have shown Lm specifically in splenic CD8a(+) DCs shortly after intravenous infection. We examined gene expression profiles of mouse DCs exposed to Lm to elucidate the molecular mechanisms underlying DCs interaction with Lm. Using a functional genomics approach, we found that Lm infection induced a cluster of late response genes including type I IFNs and interferon responsive genes (IRGs) in DCs. Type I INFs were produced at the maximal level only at 24 h post infection indicating that the regulation of IFNs in the context of Lm infection is delayed compared to the rapid response observed with viral pathogens. We showed that during Lm infection, IFNγ production and cytotoxic activity were severely impaired in NK cells compared to E. coli infection. These defects were restored by providing an exogenous source of IFNβ during the initial phase of bacterial challenge. Moreover, when treated with IFNβ during early infection, NK cells were able to reduce bacterial titer in the spleen and significantly improve survival of infected mice. These findings show that the timing of IFNβ production is fundamental to the efficient control of the bacterium during the early innate phase of Lm infection.     

Sensitivity of IFN-γ Release Assay to Detect Latent Tuberculosis Infection Is Retained in HIV-Infected Patients but Dependent on HIV/AIDS Progression

Background

Detection and treatment of latent TB infection (LTBI) in HIV infected individuals is strongly recommended to decrease morbidity and mortality in countries with high levels of HIV.

Objective

To assess the validity of a newly developed in-house ELISPOT interferon-γ release assay (IGRA) for the detection of LTBI amongst HIV infected individuals, in comparison with the Tuberculin Skin Test (TST).

Methodology/Principal Findings

ESAT6/CFP10 (EC) ELISPOT assays were performed, together with a TST, in 285 HIV infected individuals recruited in HIV clinics in Dakar, Senegal, who had no signs of active TB at time of enrolment. Thirty eight of the subjects (13.3%) failed to respond to PHA stimulation and were excluded from the analysis. In the 247 remaining patients, response to PHA did not vary according to CD4 cell count categories (p = 0.51). EC ELISPOT was positive in 125 (50.6%) subjects, while 53 (21.5%) had a positive TST. Concordance between EC ELISPOT and TST was observed in 151 patients (61.1%) (kappa = 0.23). The proportion of subjects with a positive response to the EC ELISPOT assay decreased with declining CD4 counts (p trend = 0.001), but were consistently higher than the proportion of TST responders. In multivariate analysis, the risk of being EC-ELISPOT positive in HIV infected individuals was associated with age, CD4 count and HIV-1 strain.

Conclusion

Our study indicates that IGRAs using M. tuberculosis specific antigens are likely to retain their validity for the diagnosis of LTBI among HIV positive individuals, but may be impaired by T-cell anergy in severely immuno-suppressed individuals.     

Regulation of Stomatal Tropism and Infection by Light in Cercospora zeae-maydis: Evidence for Coordinated Host/Pathogen Responses to Photoperiod?

Cercospora zeae-maydis causes gray leaf spot of maize, which has become one of the most widespread and destructive diseases of maize in the world. C. zeae-maydis infects leaves through stomata, which is predicated on the ability of the pathogen to perceive stomata and reorient growth accordingly. In this study, the discovery that light was required for C. zeae-maydis to perceive stomata and infect leaves led to the identification of CRP1, a gene encoding a putative blue-light photoreceptor homologous to White Collar-1 (WC-1) of Neurospora crassa. Disrupting CRP1 via homologous recombination revealed roles in multiple aspects of pathogenesis, including tropism of hyphae to stomata, the formation of appressoria, conidiation, and the biosynthesis of cercosporin. CRP1 was also required for photoreactivation after lethal doses of UV exposure. Intriguingly, putative orthologs of CRP1 are central regulators of circadian clocks in other filamentous fungi, raising the possibility that C. zeae-maydis uses light as a key environmental input to coordinate pathogenesis with maize photoperiodic responses. This study identified a novel molecular mechanism underlying stomatal tropism in a foliar fungal pathogen, provides specific insight into how light regulates pathogenesis in C. zeae-maydis, and establishes a genetic framework for the molecular dissection of infection via stomata and the integration of host and pathogen responses to photoperiod.     

In Vitro Immunomodulation of a Whole Blood IFN-γ Release Assay Enhances T Cell Responses in Subjects with Latent Tuberculosis Infection

Background

Activation of innate immunity via pathogen recognition receptors (PRR) modulates adaptive immune responses. PRR ligands are being exploited as vaccine adjuvants and as therapeutics, but their utility in diagnostics has not been explored. Interferon-gamma (IFN-γ) release assays (IGRAs) are functional T cell assays used to diagnose latent tuberculosis infection (LTBI); however, novel approaches are needed to improve their sensitivity.

Methods

In vitro immunomodulation of a whole blood IGRA (QuantiFERON®-TB GOLD In-Tube) with Toll-like receptor agonists poly(I:C), LPS, and imiquimod was performed on blood from subjects with LTBI and negative controls.

Results

In vitro immunomodulation significantly enhanced the response of T cells stimulated with M. tuberculosis antigens from subjects with LTBI but not from uninfected controls. Immunomodulation of IGRA revealed T cell responses in subjects with LTBI whose T cells otherwise do not respond to in vitro stimulation with antigens alone. Similar to their in vivo functions, addition of poly(I:C) and LPS to whole blood induced secretion of inflammatory cytokines and IFN-α and enhanced the surface expression of antigen presenting and costimulatory molecules on antigen presenting cells.

Conclusions

In vitro immunomodulation of whole blood IGRA may be an effective strategy for enhancing the sensitivity of T cells for diagnosis of LTBI.     

IFN-γ Production to Leishmania Antigen Supplements the Leishmania Skin Test in Identifying Exposure to L. braziliensis Infection

Background

Cutaneous leishmaniasis due to L. braziliensis (CL) is characterized by a positive delayed type hypersensitivity test (DTH) leishmania skin test (LST) and high IFN-γ production to soluble leishmania antigen (SLA). The LST is used for diagnosis of CL and for identification of individuals exposed to leishmania infection but without disease. The main aim of the present study was to identify markers of exposure to L. braziliensis infection.

Methodolgy/Principal Findings

This cohort study enrolled 308 household contacts (HC) of 76 CL index cases. HC had no active or past history of leishmaniasis. For the present cross-sectional study cytokines and chemokines were determined in supernatants of whole blood culture stimulated with SLA. Of the 308 HC, 36 (11.7%) had a positive LST but in these IFN-γ was only detected in 22 (61.1%). Moreover of the 40 HC with evidence of IFN-γ production only 22 (55%) had a positive LST. A total of 54 (17.5%) of 308 HC had specific immune response to SLA. Only a moderate agreement (Kappa = 0.52; 95% CI: 0.36–0.66) was found between LST and IFN-γ production. Moreover while enhancement of CXCL10 in cultures stimulated with SLA was observed in HC with DTH+ and IFN-γ+ and in patients with IFN-γ⁺ and DTH⁻, no enhancement of this chemokine was observed in supernatants of cells of HC with DTH⁺ and IFN-γ⁻.

Conclusions/Significance

This study shows that in addition of LST, the evaluation of antigen specific IFN-γ production should be performed to determine evidence of exposure to leishmania infection. Moreover it suggests that in some HC production of IFN-γ and CXCL10 are performed by cells not involved with DTH reaction.