Inflammasome activation, NLRP3 engagement and macrophage recruitment to tumor microenvironment are all required for Salmonella antitumor effect.

Salmonella-based cancer therapies show great potential in preclinical models, but for most cases the observed antitumor effect is transient. Understanding the basis of the antitumor efficacy might guide the design of improved strains that elicit long-lasting effects, paving the wave for clinical use.  Here, we deepened into the role of macrophages and inflammasome activation in the context of Salmonella anti-melanoma effect. We showed inflammasome activation in melanoma cells upon infection, which correlated with cell surface exposure of gasdermin-D (GSDM-D) and calreticulin (CRT) and High mobility group box 1 protein (HMGB-1) release, suggesting immunogenic cell death, particularly pyroptosis. Salmonella infection upregulated levels of Caspase-11 (Casp11) mRNA, but not Nlrp3 or Nlrc4 mRNA, the only described inflammasome receptors engaged by Salmonella, suggesting that non-canonical inflammasome activation could be occurring in melanoma cells. Intratumoral administration of Salmonella to melanoma-bearing mice elicited local inflammasome activation and interleukin-1ß (IL-1ß) production together with tumor growth retardation and extended survival in wild type but not Caspase-1/11 (Casp1/11) knockout mice despite similar levels of intratumoral IL-1ß in the later. Salmonella antitumor activity was also suppressed in melanoma bearing Nlrp3 knockout mice. Salmonella induced macrophage recruitment to the tumor site and infiltrating cells exhibited inflammasome activation. Depletion experiments confirmed that macrophages are also essential for Salmonella anti-melanoma effect. Intratumoral macrophages showed a marked M2/M1 shift soon after treatment but this inflammatory profile is then lost, which could explain the transient effect of therapy.  All in all, our results highlight CASP-1/11 axis and macrophages as essential players in Salmonella-based cancer immunotherapy and suggest a possible target for future interventions.

Polyvalent Bacterial Lysate Protects Against Pneumonia Independently of Neutrophils, IL-17A or Caspase-1 Activation.

Polyvalent bacterial lysates have been in use for decades for prevention and treatment of respiratory infections with reported clinical benefits. However, besides claims of broad immune activation, the mode of action is still a matter of debate. The lysates, formulated with the main bacterial species involved in respiratory infections, are commonly prepared by chemical or mechanical disruption of bacterial cells, what is believed influences the biological activity of the product. Here, we prepared two polyvalent lysates with the same composition but different method of bacterial cell disruption and evaluated their biological activity in a comparative fashion. We found that both bacterial lysates induce NF-kB activation in a MyD88 dependent manner, suggesting they work as TLR agonists. Further, we found that a single intranasal dose of any of the two lysates, is sufficient to protect against pneumococcal pneumonia, suggesting that they exert similar biological activity. We have previously shown that protection against pneumococcal pneumonia can also be induced by prior S. pneumoniae sub lethal infection or therapeutic treatment with a TLR5 agonist. Protection in those cases depends on neutrophil recruitment to the lungs, and can be associated with increased local expression of IL-17A. Here, we show that bacterial lysates exert protection against pneumococcal pneumonia independently of neutrophils, IL-17A or Caspase-1/11 activation, suggesting the existence of redundant mechanisms of protection. Trypsin-treated lysates afford protection to the same extent, suggesting that just small peptides suffice to exert the protective effect or that the molecules responsible for the protective effect are not proteins. Understanding the mechanism of action of bacterial lysates and deciphering the active components shall allow redesigning them with more precisely defined formulations and expanding their range of action.

Salmonella Typhimurium Triggers Extracellular Traps Release in Murine Macrophages.

Salmonella comprises two species and more than 2500 serovars with marked differences in host specificity, and is responsible for a wide spectrum of diseases, ranging from localized gastroenteritis to severe life-threatening invasive disease. The initiation of the host inflammatory response, triggered by many Pathogen-Associated Molecular Patterns (PAMPs) that Salmonella possesses, recruits innate immune cells in order to restrain the infection at the local site. Neutrophils are known for killing bacteria through oxidative burst, amid other mechanisms. Amongst those mechanisms for controlling bacteria, the release of Extracellular Traps (ETs) represents a newly described pathway of programmed cell death known as ETosis. Particularly, Neutrophil Extracellular Traps (NETs) were first described in 2004 and since then, a number of reports have demonstrated their role as a novel defense mechanism against different pathogens. This released net-like material is composed of cellular DNA decorated with histones and cellular proteins. These structures have shown ability to trap, neutralize and kill different kinds of microorganisms, ranging from viruses and bacteria to fungi and parasites. Salmonella was one of the first microorganisms that were reported to be killed by NETs and several studies have confirmed the observation and deepened into its variants. Nevertheless, much less is known about their counterparts in other immune cells, e.g. Macrophage Extracellular Traps (METs) and Salmonella-induced MET release has never been reported so far. In this work, we observed the production of METs induced by Salmonella enterica serovar Typhimurium and recorded their effect on bacteria, showing for the first time that macrophages can also release extracellular DNA traps upon encounter with Salmonella Typhimurium. Additionally we show that METs effectively immobilize and reduce Salmonella survival in a few minutes, suggesting METs as a novel immune-mediated defense mechanism against Salmonella infection. Of note, this phenomenon was confirmed in primary macrophages, since MET release was also observed in bone marrow-derived macrophages infected with Salmonella. The evidence of this peculiar mechanism provides new incipient insights into macrophages´ role against Salmonella infection and can help to design new strategies for the clinical control of this transcendental pathogen.

Publisher Correction: Comparative genomics of Salmonella enterica serovar Enteritidis ST-11 isolated in Uruguay reveals lineages associated with particular epidemiological traits.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comparative genomics of Salmonella enterica serovar Enteritidis ST-11 isolated in Uruguay reveals lineages associated with particular epidemiological traits.

Salmonella enterica serovar Enteritidis is a major cause of foodborne disease in Uruguay since 1995. We used a genomic approach to study a set of isolates from different sources and years. Whole genome phylogeny showed that most of the strains are distributed in two major lineages (E1 and E2), both belonging to MLST sequence type 11 the major ST among serovar Enteritidis. Strikingly, E2 isolates are over-represented in periods of outbreak abundance in Uruguay, while E1 span all epidemic periods. Both lineages circulate in neighbor countries at the same timescale as in Uruguay, and are present in minor numbers in distant countries. We identified allelic variants associated with each lineage. Three genes, ycdX, pduD and hsdM, have distinctive variants in E1 that may result in defective products. Another four genes (ybiO, yiaN, aas, aceA) present variants specific for the E2 lineage. Overall this work shows that S. enterica serovar Enteritidis strains circulating in Uruguay have the same phylogenetic profile than strains circulating in the region, as well as in more distant countries. Based on these results we hypothesize that the E2 lineage, which is more prevalent during epidemics, exhibits a combination of allelic variants that could be associated with its epidemic ability.

Draft Genome Sequences of Two Multidrug-Resistant Salmonella enterica Serovar Typhimurium Clinical Isolates from Uruguay.

Multidrug-resistant Salmonella enterica isolates are an increasing problem worldwide; nevertheless, the mechanisms responsible for such resistance are rarely well defined. Multidrug-resistant S. enterica serovar Typhimurium isolates ST3224 and ST827 were collected from two patients. The characteristics of both genomes and antimicrobial resistance genes were determined using next-generation sequencing.

Quillaja brasiliensis saponin-based nanoparticulate adjuvants are capable of triggering early immune responses.

Commercially available saponins are extracted from Quillaja saponaria barks, being Quil A® the most widely used. Nanoparticulate immunostimulating complexes (ISCOMs or ISCOMATRIX) formulated with these, are able to stimulate strong humoral and cellular immune responses. Recently, we formulated novel ISCOMs replacing QuilA® by QB-90 (IQB-90), a Quillaja brasiliensis leaf-extracted saponin fraction, and reported that IQB-90 improved antigen uptake, and induced systemic and mucosal antibody production, and T-cell responses. However, its mechanism of action remains unclear. In this study we provide a deeper insight into the immune stimulatory properties of QB-90 and ISCOMATRIX-like based on this fraction (IMXQB-90). We show herein that, when used as a viral vaccine adjuvant, QB-90 promotes an "immunocompetent environment". In addition, QB-90 and IMXQB-90 induce immune-cells recruitment at draining-lymph nodes and spleen. Subsequently, we prove that QB-90 or IMXQB-90 stimulated dendritic cells secret IL-1ß by mechanisms involving Caspase-1/11 and MyD88 pathways, implying canonical inflammasome activation. Finally, both formulations induce a change in the expression of cytokines and chemokines coding genes, many of which are up-regulated. Findings reported here provide important insights into the molecular and cellular mechanisms underlying the adjuvant activity of Q. brasiliensis leaf-saponins and its respective nanoparticles.

A novel prophage identified in strains from Salmonella enterica serovar Enteritidis is a phylogenetic signature of the lineage ST-1974.

Salmonella enterica serovar Enteritidis is a major agent of foodborne diseases worldwide. In Uruguay, this serovar was almost negligible until the mid 1990s but since then it has become the most prevalent. Previously, we characterized a collection of strains isolated from 1988 to 2005 and found that the two oldest strains were the most genetically divergent. In order to further characterize these strains, we sequenced and annotated eight genomes including those of the two oldest isolates. We report on the identification and characterization of a novel 44 kbp Salmonella prophage found exclusively in these two genomes. Sequence analysis reveals that the prophage is a mosaic, with homologous regions in different Salmonella prophages. It contains 60 coding sequences, including two genes, gogB and sseK3, involved in virulence and modulation of host immune response. Analysis of serovar Enteritidis genomes available in public databases confirmed that this prophage is absent in most of them, with the exception of a group of 154 genomes. All 154 strains carrying this prophage belong to the same sequence type (ST-1974), suggesting that its acquisition occurred in a common ancestor. We tested this by phylogenetic analysis of 203 genomes representative of the intraserovar diversity. The ST-1974 forms a distinctive monophyletic lineage, and the newly described prophage is a phylogenetic signature of this lineage that could be used as a molecular marker. The phylogenetic analysis also shows that the major ST (ST-11) is polyphyletic and might have given rise to almost all other STs, including ST-1974.

TLR7 agonist in combination with Salmonella as an effective antimelanoma immunotherapy.

AIM: We evaluated a novel approach combining the use of attenuated Salmonella immunotherapy with a Toll-like receptor agonist, imiquimod, in B16F1 melanoma-bearing mice. MATERIALS & METHODS: B16F1 melanoma-bearing mice were daily treated with topical imiquimod in combination with one intratumoral injection of attenuated Salmonella enterica serovar Typhimurium LVR01. RESULTS: The combined therapy resulted in retarded tumor growth and prolonged survival. Combination treatment led to an enhancement in the expression of pro-inflammatory cytokines and chemokines in the tumor microenvironment, with a Th1-skewed profile, resulting in a broad antitumor response. The induced immunity was effective in controlling the occurrence of metastasis. CONCLUSION: Salmonella LVR01 immunotherapy in combination with imiquimod is a novel approach that could be considered as an effective antimelanoma therapy.

Intranasal administration of a polyvalent bacterial lysate induces self-restricted inflammation in the lungs and a Th1/Th17 memory signature.

Respiratory tract infections are among the most frequent infections in humans causing millions of deaths especially in children and the elderly. Antibiotics and vaccines are the main available tools of control, but resistant strains are continuously arising and available vaccines only account for few of many pathogens involved. Non-specific immunotherapies are an emerging alternative to induce protective immunity at the airways. Mucosally administered polyvalent bacterial lysates (PBLs) have been widely used for decades for prevention of respiratory diseases, but the bases of their proposed therapeutic effectiveness are still controversial. Here, we show that PBL engages a pro-inflammatory gene expression program in macrophages and epithelial cells, induces an acute lung inflammatory response and elicits full protection against pneumococcal pneumonia. Chronic lung inflammation may have pathological consequences, so the capacity to regain local homeostasis after treatment is central. We found that local inflammation is fully resolved and 30 days after treatment lungs become undistinguishable from naïve mice. Nevertheless, this process leaves an immunological imprinting with a Th1/Th17 memory phenotype that may be a marker of the protective immunity elicited. Increasing our understanding of the mechanism of action of PBLs may improve the efficiency of these immunotherapies and expand their range of action.

Repression of flagella is a common trait in field isolates of Salmonella enterica serovar Dublin and is associated with invasive human infections.

The nontyphoidal Salmonella enterica serovar Dublin is adapted to cattle but infrequently infects humans, very often resulting in invasive infections with high levels of morbidity and mortality. A Salmonella-induced intestinal acute inflammatory response is postulated as a mechanism to prevent bacterial dissemination to systemic sites. In S. enterica serovar Typhimurium, flagella contribute to this response by providing motility and FliC-mediated activation of pattern recognition receptors. In this study, we found 4 Salmonella enterica isolates, with the antigenic formula 9,12:-:-, that, based on fliC sequence and multilocus sequence type (MLST) analyses, are aflagellate S. Dublin isolates. Interestingly, all were obtained from human bloodstream infections. Thus, we investigated the potential role of flagella in the unusual invasiveness exhibited by S. Dublin in humans by analyzing flagellation and proinflammatory properties of a collection of 10 S. Dublin human clinical isolates. We found that 4 of 7 blood isolates were aflagellate due to significantly reduced levels of fliC expression, whereas all 3 isolates from other sources were flagellated. Lack of flagella correlated with a reduced ability of triggering interleukin-8 (IL-8) and CCL20 chemokine expression in human intestinal Caco-2 cells and with reduced early inflammation in the ceca of streptomycin-pretreated C57/BL6 mice. These results indicate that flagella contribute to the host intestinal inflammatory response to Salmonella serovar Dublin and suggest that their absence may contribute to its systemic dissemination through dampening of the gut immune response. Analysis of FliC production in a collection of cattle isolates indicated that the aflagellate phenotype is widely distributed in field isolates of S. Dublin.

Generation and selection of anti-flagellin monoclonal antibodies useful for serotyping Salmonella enterica.

In developing countries, bacterial acute gastroenteritis continues to be an important cause of morbidity and mortality among young children. Salmonellosis constitutes a major cause of infectious enteritis worldwide, most of them associated to the consumption of contaminated food products. Traditionally, Salmonella has been classified in serovars based on varieties of O and H surface antigens. In the present work we generated and characterized a panel of anti-flagellin monoclonal antibodies (MAbs) in order to select antibodies useful for detecting the H surface antigen. Four different MAbs were obtained by somatic hybridization of splenocytes. We found two MAbs that recognised regions of flagellin conserved among different Salmonella serovars. Other two MAbs recognised structures restricted to Salmonella enterica sv. Typhimurium, being one of them suitable for agglutination tests. Using a diverse panel of S. enterica serovars with different H antigen varieties we confirmed that this MAb agglutinates specifically S. Typhimurium (antigenic formula: 4,12:i:1,2) or other serovars expressing flagellar factor i. In conclusion, we generated a valuable immunochemical tool to be used in simple assays for serotyping of epidemiologically relevant strains. The capacity to characterize specific strains and determine the primary sources of Salmonella contamination generate valuable information of the epidemiology of this microorganism, contributing to the improvement of public health.

Innate immune responses to Proteus mirabilis flagellin in the urinary tract.

Flagella are bacterial virulence factors allowing microorganisms to move over surfaces. Flagellin, the structural component of flagella, is sensed by the host via Toll and NOD-like receptors and triggers pro-inflammatory responses. The use of Toll-like receptors agonists to modulate innate immune responses has aroused great interest as an alternative to improve the treatment of diverse infectious diseases. Proteus mirabilis is a Gram negative bacterium that causes urinary tract infections in humans. In the present work we used different approaches to study the ability of P. mirabilis flagellin to induce an innate immune response. We demonstrated that P. mirabilis flagellin has the ability to induce pro-inflammatory chemokines expression in T24 bladder cultures cells and in the mouse bladder after instillation. It was evidenced also that flagellin from different P. mirabilis strains differed in their capacity to induce an innate immune response in the CacoCCL20-Luc system. Also, flagellin elicited inflammation, with recruitment of leukocytes to the bladder epithelium. Flagellin instillation before an experimental P. mirabilis infection showed that the inflammatory response due to flagellin did not help to clear the infection but favored bacterial colonization. Thus, induction of inflammatory response in the bladder did not contribute to P. mirabilis infection neutralization.

Early peritoneal immune response during Echinococcus granulosus establishment displays a biphasic behavior.

BACKGROUND: Cystic echinococcosis is a worldwide distributed helminth zoonosis caused by the larval stage of Echinococcus granulosus. Human secondary cystic echinococcosis is caused by dissemination of protoscoleces after accidental rupture of fertile cysts and is due to protoscoleces ability to develop into new metacestodes. In the experimental model of secondary cystic echinococcosis mice react against protoscoleces producing inefficient immune responses, allowing parasites to develop into cysts. Although the chronic phase of infection has been analyzed in depth, early immune responses at the site of infection establishment, e.g., peritoneal cavity, have not been well studied. Because during early stages of infection parasites are thought to be more susceptible to immune attack, this work focused on the study of cellular and molecular events triggered early in the peritoneal cavity of infected mice. PRINCIPAL FINDINGS: Data obtained showed disparate behaviors among subpopulations within the peritoneal lymphoid compartment. Regarding B cells, there is an active molecular process of plasma cell differentiation accompanied by significant local production of specific IgM and IgG2b antibodies. In addition, peritoneal NK cells showed a rapid increase with a significant percentage of activated cells. Peritoneal T cells showed a substantial increase, with predominance in CD4(+) T lymphocytes. There was also a local increase in Treg cells. Finally, cytokine response showed local biphasic kinetics: an early predominant induction of Th1-type cytokines (IFN-γ, IL-2 and IL-15), followed by a shift toward a Th2-type profile (IL-4, IL-5, IL-6, IL-10 and IL-13). CONCLUSIONS: Results reported here open new ways to investigate the involvement of immune effectors players in E. granulosus establishment, and also in the sequential promotion of Th1- toward Th2-type responses in experimental secondary cystic echinococcosis. These data would be relevant for designing rational therapies based on stimulation of effective responses and blockade of evasion mechanisms.

Live attenuated Salmonella as a vector for oral cytokine gene therapy in melanoma.

Systemic administration of cytokines has shown therapeutic benefits in cancer patients; however, serious adverse effects associated with direct protein administration prevent the wide use of this approach. We have assessed the capacity of live attenuated Salmonella to act as a vector for oral cytokine-gene therapy. Salmonella orally administered to melanoma-bearing mice was found to accumulate within the tumor, reaching up to 10(5) bacteria per gram of tumor by day 21 after bacterial inoculation. Numbers of bacteria recovered from tumor did not differ from those recovered from liver or spleen at any time point. Recombinant bacteria carrying eukaryotic expression vectors encoding the murine IL-4 or IL-18 genes were administered to groups of mice with established subcutaneous melanoma tumors. We found that a single oral dose of Salmonella carrying any of the cytokine-encoding plasmids resulted in significantly increased survival time, as compared with mice that received Salmonella carrying the parental plasmid or PBS. Increased levels of IFNgamma were found in sera of animals receiving either of the cytokine-encoding bacteria, but not in mice receiving Salmonella alone or PBS. Co-administration of both recombinant bacteria maximized the production of IFNgamma. Overall these results suggest that cytokine-encoding Salmonella can be an effective and safer alternative to systemic administration of cytokines for immunotherapy of cancer.