The Immune Inhibitor A1 Protease of Bacillus anthracis

Bacillus anthracis, an organism ubiquitous in the soil and the causative agent of anthrax, utilizes multiple mechanisms to regulate secreted factors; one example is the activity of secreted proteases. One of the most abundant proteins in the culture supernates of B. anthracis is the Immune Inhibitor A1 (InhA1) protease. Here, I demonstrate that InhA1 modulates the abundance of approximately half of the proteins secreted into the culture supernates, including substrates that are known to contribute to the ability of the organism to cause virulence. For example, InhA1 cleaves the anthrax toxin proteins, PA, LF, and EF. InhA1 also targets a number of additional proteases, including Npr599, contributing to a complex proteolytic regulatory cascade with far-reaching affects on the secretome. Using an intra-tracheal mouse model of infection, I found that an inhA-null strain is attenuated in relation to the parent strain. The data indicate that reduced virulence of the inhA mutant strain may be the result of toxin protein deregulation, decreased association with macrophages, and/or the inability to degrade host antimicrobial peptides. Given the significant modulation of the secretome by InhA1, it is likely that expression of the protease is tightly regulated. To test this I examined inhA1 transcript and protein levels in the parent and various isogenic mutant strains and found that InhA1 expression is regulated by several mechanisms. First, the steady state levels of inhA1 transcript are controlled by the regulatory protein SinR, which inhibits inhA1 expression. Second, InhA1 abundance is inversely proportional to the SinR-regulated protease camelysin, indicating the post-transcriptional regulation of InhA1 by camelysin. Third, InhA1 activity is dependent on a conserved zinc binding motif, suggesting that zinc availability regulates InhA1 activity. The convergence of these regulatory mechanisms signifies the importance of tight regulation of InhA1 activity, activity that substantially affects how B. anthracis interacts with its environment.

The Immune System of Spiders

Spiders, as all other arthropods, have an open circulatory system, and their body fluid, the hemolymph, freely moves between lymphatic vessels and the body cavities (see Wirkner and Huckstorf 2013). The hemolymph can be considered as a multifunctional organ, central for locomotion (Kropf 2013), respiration (Burmester 2013) and nutrition, and it amounts to approximately 20 % of a spider’s body weight. Any injury includes not only immediate hemolymph loss but also pathogen attacks and subsequent infections. Therefore spiders have to react to injuries in a combined manner to stop fluid loss and to defend against microbial invaders. This is achieved by an innate immune system which involves several host defence systems such as hemolymph coagulation and the production of a variety of defensive substances (Fukuzawa et al.2008). In spiders, the immune system is localised in hemocytes which are derived from the myocardium cells of the heart wall where they are produced as prohemocytes and from where they are released as different cell types into the hemolymph (Seitz 1972). They contribute to the defence against pathogens by phagocytosis, nodulation and encapsulation of invaders. The humoral response includes mechanisms which induce melanin production to destroy pathogens, a clotting cascade to stop hemolymph loss and the constitutive production of several types of antimicrobial peptides, which are stored in hemocyte granules and released into the hemolymph (Fukuzawa et al.2008) (Fig.7.1). The immune system of spiders is an innate immune system. It is hemolymph-based and characterised by a broad but not very particular specificity. Its advantage is a fast response within minutes to a few hours. This is in contrast to the adaptive immune system of vertebrates which can react to very specific pathogens, thus resulting in much more specific responses. Moreover, it creates an immunological memory during the lifetime of the species. The disadvantage is that it needs more time to react with antibody production, usually many hours to a few days, and needs to be built up during early ontogenesis.

IMMUNE MODULATION OF THE MYCOBACTERIUM TUBERCULOSIS GRANULOMATOUS RESPONSE

Tuberculosis (TB) remains a major public health burden. The immunocompetant host responds to Mycobacterium tuberculosis (MTB) infection by the formation of granulomas, which initially prevent uncontrolled bacterial proliferation and dissemination. However, increasing evidence suggests that granuloma formation promotes persistence of the organism by physically separating infected cells from effector lymphocytes and by inducing a state of non-replicating persistence in the bacilli, making them resistant to the action of antibiotics. Additionally, immune-mediated tissue destruction likely facilitates disease transmission. The granulomatous response is in part due to mycobacterial glycolipid antigens. Therefore, studies were first undertaken to determine the innate mechanisms of mycobacterial cord factor trehalose-6’6-dimycolate (TDM) on granuloma formation. Investigations using knock-out mice suggest that TNF-a is involved in the initiation of the granulomatous response, complement factor C5a generates granuloma cohesiveness, and IL-6 is necessary for maintenance of an established granulomatous responses. Studies were next performed to determine the ability of lactoferrin to modulate the immune response and pathology to mycobacterial cord factor. Lactoferrin is an iron-binding glycoprotein with immunomodulatory properties that decrease tissue damage and promote Th1 responses. Mice challenged with TDM and treated with lactoferrin had decreased size and numbers of granulomas at the peak of the granulomatous response, accompanied by increased IL-10 and TGF-b production. Finally, the ability of lactoferrin to serve as a novel therapeutic for the treatment of TB was performed by aerosol challenging mice with MTB and treating them with lactoferrin added to the drinking water. Mice given tap water had lung log10 CFUs of 7.5 ± 0.3 at week 3 post-infection. Lung CFUs were significantly decreased in mice given lactoferrin starting the day of infection (6.4 ± 0.7) and mice started therapeutically on lactoferrin at day 7 after established infection (6.5 ± 0.4). Total lung inflammation in lactoferrin treated mice was significantly decreased, with fewer areas of macrophages, increased total lymphocytes, and increased numbers of CD4+ and CD8+ cells. The lungs of lactoferrin treated mice had increased CD4+ IFN-g+ cells and IL-17 producing cells on ELISpot analysis. It is hypothesized that lactoferrin decreases bacterial burden during MTB infection by early induction of Th1 responses.

Immune response to regressor and progressor tumor variants

Most skin cancers induced in mice by Ultraviolet (UV) radiation express highly immunogenic Tumor specific transplantation antigens (TSTAs) and thus exhibit a regressor phenotype. In this study, I have used cloned genes encoding tumor antigens and oncogenes in conjunction with DNA transfection technique to isolate and characterize regressor variants from progressor tumors and vice versa. The purpose of this study was (1) to determine whether the product of a cloned gene (216) from UV-1591 tumor, which encodes a novel MHC class I antigen can function as a tumor rejection antigen when expressed on unrelated, nonantigenic, murine tumor cells or whether its function is restricted to UV-induced tumors, and (2) to determine the processes by which progressor variants derived from a regressor UV-2240 cell line by transfection with an activated Ha-ras oncogene escape the immune defenses of the normal immunocompetent host.^ To answer the first question, a spontaneously transformed, nonimmunogenic cell line (10T-1) was cotransfected with DNA from p216 and pSV2-neo plasmids. Results demonstrate that the product of a cloned TSTA gene from a UV-induced murine tumor is capable of functioning as a tumor rejection antigen when expressed on unrelated, nonantigenic tumor cells. In addition, these results indicate that this approach could be used to augment the immune response against poorly antigenic tumors.^ To answer the second question, progressor variants were isolated from a highly antigenic UV radiation-induced C3H mouse regressor fibrosarcoma cell line, UV-2240, by transfection with an activated Ha-ras oncogene. Subcutaneous injection of Ha-ras-transfected UV-2240 cells into immunocompetent C3H mice produced tumors in 4 of 36 animals. In addition, the Ha-ras-induced progressor variants produced experimental lung metastasis in both normal C3H and nude mice, although they induced more lung nodules in nude mice than in normal C3H mice. Results indicate that the progressor phenotype of the Ha-ras-induced tumor variants is not due to loss of TSTAs or MHC class I antigens. This implies that some tumors can escape the immune defenses of the normal immunocompetent host by mechanisms other than the loss of TSTAs and MHC class I antigens. (Abstract shortened with permission of author.) ^

Highly pathogenic adapted HIV-1 strains limit host immunity and dictate rapid disease progression.

OBJECTIVE: The study of HIV-1 rapid progressors has been limited to specific case reports. Nevertheless, identification and characterization of the viral and host factors involved in rapid progression are crucial when attempting to uncover the correlates of rapid disease outcome. DESIGN: We carried out comparative functional analyses in rapid progressors (n = 46) and standard progressors (n = 46) early after HIV-1 seroconversion (≤1 year). The viral traits tested were viral replicative capacity, co-receptor usage, and genomic variation. Host CD8 T-cell responses, humoral activity, and HLA immunogenetic markers were also determined. RESULTS: Our data demonstrate an unusual convergence of highly pathogenic HIV-1 strains in rapid progressors. Compared with standard progressors, rapid progressor viral strains show higher in-vitro replicative capacity (81.5 vs. 67.9%; P = 0.025) and greater X4/DM co-receptor usage (26.3 vs. 2.8%; P = 0.006) in early infection. Limited or absent functional HIV-1 CD8 T-cell responses and neutralizing activity were measured in rapid progressors. Moreover, the increase in common HLA allele-restricted CD8 T-cell escape mutations in rapid progressors acts as a signature of uncontrolled HIV-1 replication and early impairment of adaptive cellular responses. CONCLUSION: Our data support a dominant role for viral factors in rapid progressors. Robust HIV-1 replication and intrinsic viral properties limit host adaptive immune responses, thus driving rapid disease progression.

Matrix metalloproteinases: multifunctional effectors of inflammation in multiple sclerosis and bacterial meningitis

Matrix metalloproteinases (MMPs) are a family of Zn2+-dependent endopeptidases targeting extracellular matrix (ECM) compounds as well as a number of other proteins. Their proteolytic activity acts as an effector mechanism of tissue remodeling in physiologic and pathologic conditions, and as modulator of inflammation. In the context of neuro-inflammatory diseases, MMPs have been implicated in processes such as (a) blood-brain barrier (BBB) and blood-nerve barrier opening, (b) invasion of neural tissue by blood-derived immune cells, (c) shedding of cytokines and cytokine receptors, and (d) direct cellular damage in diseases of the peripheral and central nervous system. This review focuses on the role of MMPs in multiple sclerosis (MS) and bacterial meningitis (BM), two neuro-inflammatory diseases where current therapeutic approaches are insufficient to prevent severe disability in the majority of patients. Inhibition of enzymatic activity may prevent MMP-mediated neuronal damage due to an overactive or deviated immune response in both diseases. Downregulation of MMP release may be the molecular basis for the beneficial effect of IFN-beta and steroids in MS. Instead, synthetic MMP inhibitors offer the possibility to shut off enzymatic activity of already activated MMPs. In animal models of MS and BM, they efficiently attenuated clinical disease symptoms and prevented brain damage due to excessive metalloproteinase activity. However, the required target profile for the therapeutic use of this novel group of compounds in human disease is not yet sufficiently defined and may be different depending on the type and stage of disease. Currently available MMP inhibitors show little target-specificity within the MMP family and may lead to side-effects due to interference with physiological functions of MMPs. Results from human MS and BM indicate that only a restricted number of MMPs specific for each disease is up-regulated. MMP inhibitors with selective target profiles offer the possibility of a more efficient therapy of MS and BM and may enter clinical trials in the near future.

The Host Nonsense-Mediated mRNA Decay Pathway Restricts Mammalian RNA Virus Replication

In addition to classically defined immune mechanisms, cell-intrinsic processes can restrict virus infection and have shaped virus evolution. The details of this virus-host interaction are still emerging. Following a genome-wide siRNA screen for host factors affecting replication of Semliki Forest virus (SFV), a positive-strand RNA (+RNA) virus, we found that depletion of nonsense-mediated mRNA decay (NMD) pathway components Upf1, Smg5, and Smg7 led to increased levels of viral proteins and RNA and higher titers of released virus. The inhibitory effect of NMD was stronger when virus replication efficiency was impaired by mutations or deletions in the replicase proteins. Consequently, depletion of NMD components resulted in a more than 20-fold increase in production of these attenuated viruses. These findings indicate that a cellular mRNA quality control mechanism serves as an intrinsic barrier to the translation of early viral proteins and the amplification of +RNA viruses in animal cells.

Potent innate immune response to pathogenic leptospira in human whole blood.

BACKGROUND Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires. METHODOLOGY/PRINCIPAL FINDINGS We investigated the host response and killing of virulent and non-virulent Leptospira of different serovars by human THP-1 cells, human PBMC's and human whole blood. Virulence of each leptospiral strain was tested in a well accepted standard guinea pig model. Virulent leptospires displayed complement resistance in human serum and whole blood while in-vitro attenuated non-virulent leptospires were rapidly killed in a complement dependent manner. In vitro stimulation of THP-1 and PBMC's with heat-killed and living leptospires showed differential serovar and cell type dependence of cytokine induction. However, at low, physiological, leptospiral dose, living virulent complement resistant strains were consistently more potent in whole blood stimulations than the corresponding non-virulent complement sensitive strains. At higher dose living virulent and non-virulent leptospires were equipotent in whole blood. Inhibition of different TLRs indicated that both TLR2 and TLR4 as well as TLR5 play a role in the whole blood cytokine response to living leptospires. CONCLUSIONS/SIGNIFICANCE Thus, in a minimally altered system as human whole blood, highly virulent Leptospira are potent inducers of the cytokine response.

The liver may act as a firewall mediating mutualism between the host and its gut commensal microbiota.

A prerequisite for establishment of mutualism between the host and the microbial community that inhabits the large intestine is the stringent mucosal compartmentalization of microorganisms. Microbe-loaded dendritic cells trafficking through lymphatics are arrested at the mesenteric lymph nodes, which constitute the firewall of the intestinal lymphatic circulation. We show in different mouse models that the liver, which receives the intestinal venous blood circulation, forms a vascular firewall that captures gut commensal bacteria entering the bloodstream during intestinal pathology. Phagocytic Kupffer cells in the liver of mice clear commensals from the systemic vasculature independently of the spleen through the liver's own arterial supply. Damage to the liver firewall in mice impairs functional clearance of commensals from blood, despite heightened innate immunity, resulting in spontaneous priming of nonmucosal immune responses through increased systemic exposure to gut commensals. Systemic immune responses consistent with increased extraintestinal commensal exposure were found in humans with liver disease (nonalcoholic steatohepatitis). The liver may act as a functional vascular firewall that clears commensals that have penetrated either intestinal or systemic vascular circuits.

The interplay between host genetic variation, viral replication and microbial translocation in untreated HIV-infected individuals.

Systemic immune activation, a major determinant of HIV disease progression, is the result of a complex interplay between viral replication, dysregulation of the immune system, and microbial translocation due to gut mucosal damage. While human genetic variants influencing HIV viral load have been identified, it is unknown to what extent the host genetic background contributes to inter-individual differences in other determinants of HIV pathogenesis like gut damage and microbial translocation. Using samples and data from 717 untreated participants in the Swiss HIV Cohort Study and a genome-wide association study design, we searched for human genetic determinants of plasma levels of intestinal fatty-acid binding protein (I-FABP/FABP2), a marker of gut damage, and of soluble sCD14 (sCD14), a marker of LPS bioactivity and microbial translocation. We also assessed the correlations between HIV viral load, sCD14 and I-FABP. While we found no genome-wide significant determinant of the tested plasma markers, we observed strong associations between sCD14 and both HIV viral load and I-FABP, shedding new light on the relationships between processes that drive progression of untreated HIV infection.

Current concepts: host-pathogen interactions in cystic fibrosis airways disease.

Chronic infection and inflammation are defining characteristics of cystic fibrosis (CF) airway disease. Conditions within the airways of patients living with CF are conducive to colonisation by a variety of opportunistic bacterial, viral and fungal pathogens. Improved molecular identification of microorganisms has begun to emphasise the polymicrobial nature of infections in the CF airway microenvironment. Changes to CF airway physiology through loss of cystic fibrosis transmembrane conductance regulator functionality result in a wide range of immune dysfunctions, which permit pathogen colonisation and persistence. This review will summarise the current understanding of how CF pathogens infect, interact with and evade the CF host.

To sense or not to sense viral RNA--essentials of coronavirus innate immune evasion.

An essential function of innate immunity is to distinguish self from non-self and receptors have evolved to specifically recognize viral components and initiate the expression of antiviral proteins to restrict viral replication. Coronaviruses are RNA viruses that replicate in the host cytoplasm and evade innate immune sensing in most cell types, either passively by hiding their viral signatures and limiting exposure to sensors or actively, by encoding viral antagonists to counteract the effects of interferons. Since many cytoplasmic viruses exploit similar mechanisms of innate immune evasion, mechanistic insight into the direct interplay between viral RNA, viral RNA-processing enzymes, cellular sensors and antiviral proteins will be highly relevant to develop novel antiviral targets and to restrict important animal and human infections.

Developmental oestrogen exposure differentially modulates IGF-I and TNF-α expression levels in immune organs of Yersinia ruckeri-challenged young adult rainbow trout (Oncorhynchus mykiss).

Intensified aquaculture has strong impact on fish health by stress and infectious diseases and has stimulated the interest in the orchestration of cytokines and growth factors, particularly their influence by environmental factors, however, only scarce data are available on the GH/IGF-system, central physiological system for development and tissue shaping. Most recently, the capability of the host to cope with tissue damage has been postulated as critical for survival. Thus, the present study assessed the combined impacts of estrogens and bacterial infection on the insulin-like growth factors (IGF) and tumor-necrosis factor (TNF)-α. Juvenile rainbow trout were exposed to 2 different concentrations of 17β-estradiol (E2) and infected with Yersinia ruckeri. Gene expressions of IGF-I, IGF-II and TNF-α were measured in liver, head kidney and spleen and all 4 estrogen receptors (ERα1, ERα2, ERβ1 and ERβ2) known in rainbow trout were measured in liver. After 5 weeks of E2 treatment, hepatic up-regulation of ERα1 and ERα2, but down-regulation of ERß1 and ERß2 were observed in those groups receiving E2-enriched food. In liver, the results further indicate a suppressive effect of Yersinia-infection regardless of E2-treatment on day 3, but not of E2-treatment on IGF-I whilst TNF-α gene expression was not influenced by Yersinia-infection but was reduced after 5 weeks of E2-treatment. In spleen, the results show a stimulatory effect of Yersinia-infection, but not of E2-treatment on both, IGF-I and TNF-α gene expressions. In head kidney, E2 strongly suppressed both, IGF-I and TNF-α. To summarise, the treatment effects were tissue- and treatment-specific and point to a relevant role of IGF-I in infection.

The interplay between the gut microbiota and the immune system.

The impact of the gut microbiota on immune homeostasis within the gut and, importantly, also at systemic sites has gained tremendous research interest over the last few years. The intestinal microbiota is an integral component of a fascinating ecosystem that interacts with and benefits its host on several complex levels to achieve a mutualistic relationship. Host-microbial homeostasis involves appropriate immune regulation within the gut mucosa to maintain a healthy gut while preventing uncontrolled immune responses against the beneficial commensal microbiota potentially leading to chronic inflammatory bowel diseases (IBD). Furthermore, recent studies suggest that the microbiota composition might impact on the susceptibility to immune-mediated disorders such as autoimmunity and allergy. Understanding how the microbiota modulates susceptibility to these diseases is an important step toward better prevention or treatment options for such diseases.