Syphilis and the host: multi-omic analysis of host cellular responses to Treponema pallidum provides novel insight into syphilis pathogenesis.

Introduction: Syphilis is a chronic, multi-stage infection caused by the extracellular bacterium Treponema pallidum ssp. pallidum. Treponema pallidum widely disseminates through the vasculature, crosses endothelial, blood-brain and placental barriers, and establishes systemic infection. Although the capacity of T. pallidum to traverse the endothelium is well-described, the response of endothelial cells to T. pallidum exposure, and the contribution of this response to treponemal traversal, is poorly understood. Methods: To address this knowledge gap, we used quantitative proteomics and cytokine profiling to characterize endothelial responses to T. pallidum. Results: Proteomic analyses detected altered host pathways controlling extracellular matrix organization, necroptosis and cell death, and innate immune signaling. Cytokine analyses of endothelial cells exposed to T. pallidum revealed increased secretion of interleukin (IL)-6, IL-8, and vascular endothelial growth factor (VEGF), and decreased secretion of monocyte chemoattractant protein-1 (MCP-1). Discussion: This study provides insight into the molecular basis of syphilis disease symptoms and the enhanced susceptibility of individuals infected with syphilis to HIV co-infection. These investigations also enhance understanding of the host response to T. pallidum exposure and the pathogenic strategies used by T. pallidum to disseminate and persist within the host. Furthermore, our findings highlight the critical need for inclusion of appropriate controls when conducting T. pallidum-host cell interactions using in vitro- and in vivo-grown T. pallidum.

MHCII+CD80+ thymic eosinophils increase in abundance during neonatal development in mice and their accumulation is microbiota dependent.

Eosinophils are present in the thymus of mammals, yet their function at this site during homeostatic development is unknown. We used flow cytometry to determine the abundance and phenotype of eosinophils (here defined as SSchigh SiglecF+ CD11b+ CD45+ cells) in the thymus of mice during the neonatal period, the later postnatal period, and into adulthood. We show that both the total number of thymic eosinophils and their frequency among leukocytes increase over the first 2 wk of life and that their accumulation in the thymus is dependent on the presence of an intact bacterial microbiota. We report that thymic eosinophils express the interleukin-5 receptor (CD125), CD80, and IDO, and that subsets of thymic eosinophils express CD11c and major histocompatibility complex II (MHCII). We found that the frequency of MHCII-expressing thymic eosinophils increases over the first 2 wk of life, and that during this early-life period the highest frequency of MHCII-expressing thymic eosinophils is located in the inner medullary region. These data suggest a temporal and microbiota-dependent regulation of eosinophil abundance and functional capabilities in the thymus.

Identification and Functional Characterization of Peptides With Antimicrobial Activity From the Syphilis Spirochete, Treponema pallidum.

The etiological agent of syphilis, Treponema pallidum ssp. pallidum, is a highly invasive "stealth" pathogen that can evade the host immune response and persist within the host for decades. This obligate human pathogen is adept at establishing infection and surviving at sites within the host that have a multitude of competing microbes, sometimes including pathogens. One survival strategy employed by bacteria found at polymicrobial sites is elimination of competing microorganisms by production of antimicrobial peptides (AMPs). Antimicrobial peptides are low molecular weight proteins (miniproteins) that function directly via inhibition and killing of microbes and/or indirectly via modulation of the host immune response, which can facilitate immune evasion. In the current study, we used bioinformatics to show that approximately 7% of the T. pallidum proteome is comprised of miniproteins of 150 amino acids or less with unknown functions. To investigate the possibility that AMP production is an unrecognized defense strategy used by T. pallidum during infection, we developed a bioinformatics pipeline to analyze the complement of T. pallidum miniproteins of unknown function for the identification of potential AMPs. This analysis identified 45 T. pallidum AMP candidates; of these, Tp0451a and Tp0749 were subjected to further bioinformatic analyses to identify AMP critical core regions (AMPCCRs). Four potential AMPCCRs from the two predicted AMPs were identified and peptides corresponding to these AMPCCRs were experimentally confirmed to exhibit bacteriostatic and bactericidal activity against a panel of biologically relevant Gram-positive and Gram-negative bacteria. Immunomodulation assays performed under inflammatory conditions demonstrated that one of the AMPCCRs was also capable of differentially regulating expression of two pro-inflammatory chemokines [monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8)]. These findings demonstrate proof-of-concept for our developed AMP identification pipeline and are consistent with the novel concept that T. pallidum expresses AMPs to defend against competing microbes and modulate the host immune response.

Small Intestinal Levels of the Branched Short-Chain Fatty Acid Isovalerate Are Elevated during Infection with Heligmosomoides polygyrus and Can Promote Helminth Fecundity.

Heligmosomoides polygyrus is a helminth which naturally infects mice and is widely used as a laboratory model of chronic small intestinal helminth infection. While it is known that infection with H. polygyrus alters the composition of the host's bacterial microbiota, the functional implications of this alteration are unclear. We investigated the impact of H. polygyrus infection on short-chain fatty acid (SCFA) levels in the mouse intestine and sera. We found that helminth infection resulted in significantly upregulated levels of the branched SCFA isovaleric acid, exclusively in the proximal small intestine, which is the site of H. polygyrus colonization. We next set out to test the hypothesis that elevating local levels of isovaleric acid was a strategy used by H. polygyrus to promote its own fitness within the mammalian host. To test this, we supplemented the drinking water of mice with isovalerate during H. polygyrus infection and examined whether this affected helminth fecundity or chronicity. We did not find that isovaleric acid supplementation affected helminth chronicity; however, we found that it did promote helminth fecundity, as measured by helminth egg output in the feces of mice. Through antibiotic treatment of helminth-infected mice, we found that the bacterial microbiota was required in order to support elevated levels of isovaleric acid in the proximal small intestine during helminth infection. Overall, our data reveal that during H. polygyrus infection there is a microbiota-dependent localized increase in the production of isovaleric acid in the proximal small intestine and that this supports helminth fecundity in the murine host.

Impaired host resistance to Salmonella during helminth co-infection is restored by anthelmintic treatment prior to bacterial challenge.

Intestinal helminth infection can impair host resistance to co-infection with enteric bacterial pathogens. However, it is not known whether helminth drug-clearance can restore host resistance to bacterial infection. Using a mouse helminth-Salmonella co-infection system, we show that anthelmintic treatment prior to Salmonella challenge is sufficient to restore host resistance to Salmonella. The presence of the small intestine-dwelling helminth Heligmosomoides polygyrus at the point of Salmonella infection supports the initial establishment of Salmonella in the small intestinal lumen. Interestingly, if helminth drug-clearance is delayed until Salmonella has already established in the small intestinal lumen, anthelmintic treatment does not result in complete clearance of Salmonella. This suggests that while the presence of helminths supports initial Salmonella colonization, helminths are dispensable for Salmonella persistence in the host small intestine. These data contribute to the mechanistic understanding of how an ongoing or prior helminth infection can affect pathogenic bacterial colonization and persistence in the mammalian intestine.

Littermate-Controlled Experiments Reveal Eosinophils Are Not Essential for Maintaining Steady-State IgA and Demonstrate the Influence of Rearing Conditions on Antibody Phenotypes in Eosinophil-Deficient Mice.

Conflicting data has emerged regarding a role for eosinophils in IgA production, with some reports that eosinophils support both secretory and circulating IgA levels during homeostasis. Previous studies have compared antibody levels between wildtype and eosinophil-deficient mice, but these mice were obtained from different commercial vendors and/or were not littermates. Thus, the possibility remains that extrinsic environmental factors, rather than an intrinsic lack of eosinophils, are responsible for the reports of reduced IgA in eosinophil-deficient mice. Here we used wild-type and eosinophil-deficient (ΔdblGATA) mice that were purchased from a single vendor, subsequently bred in-house and either co-housed as adults, co-reared from birth or raised as littermates. We found no differences in the levels of secretory IgA or in the numbers of small intestinal IgA-producing plasma cells between wild-type and ΔdblGATA mice, demonstrating that under controlled steady-state conditions eosinophils are not essential for the maintenance of secretory IgA in the intestinal tract. While we found that levels of IgM and IgE were significantly elevated in the serum of ΔdblGATA mice compared to co-reared or co-housed wild-type mice, no significant differences in these or other circulating antibody isotypes were identified between genotypes in littermate-controlled experiments. Our results demonstrate that eosinophils are not required to maintain secretory or circulating IgA production and the absence of eosinophils does not impact circulating IgG1, IgG2b, IgM, or IgE levels during homeostasis. These findings emphasize the importance of optimally controlling rearing and housing conditions throughout life between mice of different genotypes.

Microfluidic Shear Processing Control of Biological Reduction Stimuli-Responsive Polymer Nanoparticles for Drug Delivery.

We demonstrate microfluidic manufacturing of glutathione (GSH)-responsive polymer nanoparticles (PNPs) with controlled in vitro pharmacological properties for selective drug delivery. This work leverages previous fundamental work on microfluidic control of the physicochemical properties of GSH-responsive PNPs containing cleavable disulfide groups in two different locations (core and interface, DualM PNPs). In this paper, we employ a two-phase gas-liquid microfluidic reactor for the flow-directed manufacturing of paclitaxel-loaded or DiI-loaded DualM PNPs (PAX-PNPs or DiI-PNPs, where DiI is a fluorescent drug surrogate dye). We find that both PAX-PNPs and DiI-PNPs exhibit similar flow-tunable sizes, morphologies, and internal structures to those previously described for empty DualM PNPs. Fluorescent imaging of DiI-PNP formulations shows that microfluidic manufacturing greatly improves the homogeneity of drug dispersion within the PNP population compared to standard bulk microprecipitation. Encapsulation of PAX in DualM PNPs significantly increases its selectivity to cancerous cells, with various PAX-PNP formulations showing higher cytotoxicity against cancerous MCF-7 cells than against non-cancerous HaCaT cells, in contrast to free PAX, which showed similar cytotoxicity in the two cell lines. In addition, the characterization of DualM PNP formulations formed at various microfluidic flow rates reveals that critical figures of merit for drug delivery function-including encapsulation efficiencies, GSH-triggered release rates, rates of cell uptake, cytotoxicities, and selectivity to cancerous cells-exhibit microfluidic flow tunability that mirrors trends in PNP size. These results highlight the potential of two-phase microfluidic manufacturing for controlling both structure and drug delivery function of biological stimuli-responsive nanomedicines toward improved therapeutic outcomes.

The impact of a helminth-modified microbiome on host immunity.

Intestinal helminths have well-characterized modulatory effects on mammalian immune pathways. Ongoing helminth infection has been associated with both the suppression of allergies and an altered susceptibility to microbial infections. Enteric helminths share a niche with the intestinal microbiota, and the presence of helminths alters the microbiota composition and the metabolic signature of the host. Recent studies have demonstrated that the helminth-modified intestinal microbiome has the capacity to modify host immune responses even in the absence of live helminth infection. This article discusses the mechanisms by which helminths modify the intestinal microbiome of mammals, and reviews the evidence for a helminth-modified microbiome directly influencing host immunity during infectious and inflammatory diseases. Understanding the multifaceted mechanisms that underpin helminth immunomodulation will pave the way for novel therapies to combat infectious and inflammatory diseases.

Functional specialization of intestinal dendritic cell subsets during Th2 helminth infection in mice.

Dendritic cells (DCs) are essential in dictating the nature and effectiveness of immune responses. In the intestine DCs can be separated into discrete subsets, defined by expression of CD11b and CD103, each with different developmental requirements and distinct functional potential. Recent evidence has shown that different intestinal DC subsets are involved in the induction of T helper (Th)17 and regulatory T cell responses, but the cells that initiate Th2 immune responses are still incompletely understood. We show that in the Th2 response to an intestinal helminth in mice, only CD11b+ and not CD11b- DCs accumulate in the local lymph node, upregulate PDL2 and express markers of alternative activation. An enteric Th1 response instead activated both CD11b+ and CD11b- DCs without eliciting alternative activation in either population. Functionally, only CD11b+ DCs activated during helminth infection supported Th2 differentiation in naive CD4+ T cells. Together our data demonstrate that the ability to prime Th2 cells during intestinal helminth infection, is a selective and inducible characteristic of CD11b+ DCs.

Early life factors that affect allergy development.

The incidence of allergic disease continues to rise in industrialized countries. The rapid increase in the incidence of allergic disease throughout the past half century suggests that recently altered environmental factors are driving allergy development. Accumulating evidence suggests that environmental experiences that occur during the first months of life can influence the risk of allergic sensitization. In this Review, we present the evidence relating to specific early life exposures that affect future allergy development, and discuss how these exposures may promote either tolerance or allergic sensitization.

Enteric Helminths Promote Salmonella Coinfection by Altering the Intestinal Metabolome.

Intestinal helminth infections occur predominantly in regions where exposure to enteric bacterial pathogens is also common. Helminth infections inhibit host immunity against microbial pathogens, which has largely been attributed to the induction of regulatory or type 2 (Th2) immune responses. Here we demonstrate an additional 3-way interaction in which helminth infection alters the metabolic environment of the host intestine to enhance bacterial pathogenicity. We show that an ongoing helminth infection increased colonization by Salmonella independently of T regulatory or Th2 cells. Instead, helminth infection altered the metabolic profile of the intestine, which directly enhanced bacterial expression of Salmonella pathogenicity island 1 (SPI-1) genes and increased intracellular invasion. These data reveal a novel mechanism by which a helminth-modified metabolome promotes susceptibility to bacterial coinfection.

Salmonella Rapidly Regulates Membrane Permeability To Survive Oxidative Stress.

UNLABELLED: The outer membrane (OM) of Gram-negative bacteria provides protection against toxic molecules, including reactive oxygen species (ROS). Decreased OM permeability can promote bacterial survival under harsh circumstances and protects against antibiotics. To better understand the regulation of OM permeability, we studied the real-time influx of hydrogen peroxide in Salmonella bacteria and discovered two novel mechanisms by which they rapidly control OM permeability. We found that pores in two major OM proteins, OmpA and OmpC, could be rapidly opened or closed when oxidative stress is encountered and that the underlying mechanisms rely on the formation of disulfide bonds in the periplasmic domain of OmpA and TrxA, respectively. Additionally, we found that a Salmonella mutant showing increased OM permeability was killed more effectively by treatment with antibiotics. Together, these results demonstrate that Gram-negative bacteria regulate the influx of ROS for defense against oxidative stress and reveal novel targets that can be therapeutically targeted to increase bacterial killing by conventional antibiotics. IMPORTANCE: Pathogenic bacteria have evolved ways to circumvent inflammatory immune responses. A decrease in bacterial outer membrane permeability during infection helps protect bacteria from toxic molecules produced by the host immune system and allows for effective colonization of the host. In this report, we reveal molecular mechanisms that rapidly alter outer membrane pores and their permeability in response to hydrogen peroxide and oxidative stress. These mechanisms are the first examples of pores that are rapidly opened or closed in response to reactive oxygen species. Moreover, one of these mechanisms can be targeted to artificially increase membrane permeability and thereby increase bacterial killing by the antibiotic cefotaxime during in vitro experiments and in a mouse model of infection. We envision that a better understanding of the regulation of membrane permeability will lead to new targets and treatment options for multidrug-resistant infections.

Analysis of bacterial survival after exposure to reactive oxygen species or antibiotics.

The redox balance in a variety of Gram-negative bacteria was explored using redox sensitive GFP (roGFP2), J. van der Heijden et al. doi:10.1016/j.freeradbiomed.2015.11.029[1]. This data article provides Supporting material to further investigate the relationship between Salmonella typhimurium survival and oxidative stress. The first set of data presented in this article, shows the percentage of surviving bacteria after exposure to hydrogen peroxide. The second set of data shows the concentration of hydrogen peroxide that was produced by S. Typhimurium in different growth phases. The last set of data shows the percentage of surviving S. Typhimurium bacteria after exposure to different antibiotics.

Exploring the redox balance inside gram-negative bacteria with redox-sensitive GFP.

Aerobic bacteria are continuously fighting potential oxidative stress due to endogenous and exogenous reactive oxygen species (ROS). To achieve this goal, bacteria possess a wide array of defenses and stress responses including detoxifying enzymes like catalases and peroxidases; however until now, the dynamics of the intra-bacterial redox balance remained poorly understood. Herein, we used redox-sensitive GFP (roGFP2) inside a variety of gram-negative bacteria to study real-time redox dynamics immediately after a challenge with hydrogen peroxide. Using this biosensor, we determined the individual contributions of catalases and peroxidases and found that each enzyme contributes more to rapid detoxification or to prolonged catalytic activity. We also found that the total catalytic power is affected by environmental conditions. Additionally, using a Salmonella strain that is devoid of detoxifying enzymes, we examined endogenous ROS production. By measuring endogenous ROS production, we assessed the role of oxidative stress in toxicity of heavy metals and antibiotics. We found that exposure to nickel induced significant oxidative stress whereas cobalt (which was previously implicated to induce oxidative stress) did not induce ROS formation. Since a turbulent debate evolves around oxidative stress as a general killing mechanism by antibiotics (aminoglycosides, fluoroquinolones and ß-lactams), we measured oxidative stress in bacteria that were challenged with these antibiotics. Our results revealed that antibiotics do not induce ROS formation in bacteria thereby disputing a role for oxidative stress as a general killing mechanism. Together, our results expose how the intra-bacterial redox balance in individual microorganisms is affected by environmental conditions and encounters with stress-inducing compounds. These findings demonstrate the significant potential of roGFP2 as a redox biosensor in gram-negative bacteria to investigate redox dynamics under a variety of circumstances.

Worming Their Way into the Picture: Microbiota Help Helminths Modulate Host Immunity.

Parasitic helminths are potent regulators of host immunity, including inhibition of allergic inflammation. In this issue of Immunity, Zaiss et al. (2015) reveal that microbiota compositional shifts during helminth infection contribute to the multifaceted ways that helminths modulate host immunity.

Cohabitation in the Intestine: Interactions among Helminth Parasites, Bacterial Microbiota, and Host Immunity.

Both intestinal helminth parasites and certain bacterial microbiota species have been credited with strong immunomodulatory effects. Recent studies reported that the presence of helminth infection alters the composition of the bacterial intestinal microbiota and, conversely, that the presence and composition of the bacterial microbiota affect helminth colonization and persistence within mammalian hosts. This article reviews recent findings on these reciprocal relationships, in both human populations and mouse models, at the level of potential mechanistic pathways and the implications these bear for immunomodulatory effects on allergic and autoimmune disorders. Understanding the multidirectional complex interactions among intestinal microbes, helminth parasites, and the host immune system allows for a more holistic approach when using probiotics, prebiotics, synbiotics, antibiotics, and anthelmintics, as well as when designing treatments for autoimmune and allergic conditions.

A Highly Effective Component Vaccine against Nontyphoidal Salmonella enterica Infections.

UNLABELLED: Nontyphoidal Salmonella enterica (NTS) infections are a major burden to global public health, as they lead to diseases ranging from gastroenteritis to systemic infections and there is currently no vaccine available. Here, we describe a highly effective component vaccine against S. enterica serovar Typhimurium in both gastroenteritis and systemic murine infection models. We devised an approach to generate supernatants of S. enterica serovar Typhimurium, an organism that is highly abundant in virulence factors. Immunization of mice with this supernatant resulted in dramatic protection against a challenge with serovar Typhimurium, showing increased survival in the systemic model and decreased intestinal pathology in the gastrointestinal model. Protection correlated with specific IgA and IgG levels in the serum and specific secretory IgA levels in the feces of immunized mice. Initial characterization of the protective antigens in the bacterial culture supernatants revealed a subset of antigens that exhibited remarkable stability, a highly desirable characteristic of an effective vaccine to be used under suboptimal environmental conditions in developing countries. We were able to purify a subset of the peptides present in the supernatants and show their potential for immunization of mice against serovar Typhimurium resulting in a decreased level of colonization. This component vaccine shows promise with regard to protecting against NTS, and further work should significantly help to establish vaccines against these prevalent infections. IMPORTANCE: Salmonella enterica infections other than typhoid and paratyphoid fever are a major global health burden, as they cause high morbidity and mortality worldwide. Strategies that prevent Salmonella-related diseases are greatly needed, and there is a significant push for the development of vaccines against nontyphoidal Salmonella enterica serovars. In this work, we describe an S. Typhimurium supernatant-derived vaccine that is effective in reducing bacterial colonization in mouse models of gastroenteritis as well as invasive disease. This is a component vaccine that shows high stability to heat, a feature that is important for use under suboptimal conditions, such as those found in sub-Saharan Africa.

Diet and specific microbial exposure trigger features of environmental enteropathy in a novel murine model.

Environmental enteropathy (EE) is a subclinical chronic inflammatory disease of the small intestine and has a profound impact on the persistence of childhood malnutrition worldwide. However, the aetiology of the disease remains unknown and no animal model exists to date, the creation of which would aid in understanding this complex disease. Here we demonstrate that early-life consumption of a moderately malnourished diet, in combination with iterative oral exposure to commensal Bacteroidales species and Escherichia coli, remodels the murine small intestine to resemble features of EE observed in humans. We further report the profound changes that malnutrition imparts on the small intestinal microbiota, metabolite and intraepithelial lymphocyte composition, along with the susceptibility to enteric infection. Our findings provide evidence indicating that both diet and microbes combine to contribute to the aetiology of EE, and describe a novel murine model that can be used to elucidate the mechanisms behind this understudied disease.

Concerted activity of IgG1 antibodies and IL-4/IL-25-dependent effector cells trap helminth larvae in the tissues following vaccination with defined secreted antigens, providing sterile immunity to challenge infection.

Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations.

The hygiene hypothesis: current perspectives and future therapies.

Developed countries have experienced a steady increase in atopic disease and disorders of immune dysregulation since the 1980s. This increase parallels a decrease in infectious diseases within the same time period, while developing countries seem to exhibit the opposite effect, with less immune dysregulation and a higher prevalence of infectious disease. The "hygiene hypothesis", proposed by Strachan in 1989, aimed to explain this peculiar generational rise in immune dysregulation. However, research over the past 10 years provides evidence connecting the commensal and symbiotic microbes (intestinal microbiota) and parasitic helminths with immune development, expanding the hygiene hypothesis into the "microflora" and "old friends" hypotheses, respectively. There is evidence that parasitic helminths and commensal microbial organisms co-evolved with the human immune system and that these organisms are vital in promoting normal immune development. Current research supports the potential for manipulation of the bacterial intestinal microbiota to treat and even prevent immune dysregulation in the form of atopic disease and other immune-mediated disorders (namely inflammatory bowel disease and type 1 diabetes). Both human and animal model research are crucial in understanding the mechanistic links between these intestinal microbes and helminth parasites, and the human immune system. Pro-, pre-, and synbiotic, as well as treatment with live helminth and excretory/secretory helminth product therapies, are all potential therapeutic options for the treatment and prevention of these diseases. In the future, therapeutics aimed at decreasing the prevalence of inflammatory bowel disease, type 1 diabetes, and atopic disorders will likely involve personalized microbiota and/or helminth treatments used early in life.