Guanylate-binding protein-5 is involved in inflammasome activation by bacterial DNA but only the cooperation of multiple GBPs accounts for control of Brucella abortus infection.

Introduction: Guanylate-binding proteins (GBPs) are produced in response to pro-inflammatory signals, mainly interferons. The most studied cluster of GBPs in mice is on chromosome 3. It comprises the genes for GBP1-to-3, GBP5 and GBP7. In humans, all GBPs are present in a single cluster on chromosome 1. Brucella abortus is a Gram-negative bacterium known to cause brucellosis, a debilitating disease that affects both humans and animals. Our group demonstrated previously that GBPs present on murine chromosome 3 (GBPchr3) is important to disrupt Brucella-containing vacuole and GBP5 itself is important to Brucella intracellular LPS recognition. In this work, we investigated further the role of GBPs during B. abortus infection. Methods and results: We observed that all GBPs from murine chromosome 3 are significantly upregulated in response to B. abortus infection in mouse bone marrow-derived macrophages. Of note, GBP5 presents the highest expression level in all time points evaluated. However, only GBPchr3-/- cells presented increased bacterial burden compared to wild-type macrophages. Brucella DNA is an important Pathogen-Associated Molecular Pattern that could be available for inflammasome activation after BCV disruption mediated by GBPs. In this regard, we observed reduced IL-1ß production in the absence of GBP2 or GBP5, as well as in GBPchr3-/- murine macrophages. Similar result was showed by THP-1 macrophages with downregulation of GBP2 and GBP5 mediated by siRNA. Furthermore, significant reduction on caspase-1 p20 levels, LDH release and Gasdermin-D conversion into its mature form (p30 N-terminal subunit) was observed only in GBPchr3-/- macrophages. In an in vivo perspective, we found that GBPchr3-/- mice had increased B. abortus burden and higher number of granulomas per area of liver tissue, indicating increased disease severity. Discussion/conclusion: Altogether, these results demonstrate that although GBP5 presents a high expression pattern and is involved in inflammasome activation by bacterial DNA in macrophages, the cooperation of multiple GBPs from murine chromosome 3 is necessary for full control of Brucella abortus infection.

Current Understanding of Bacillus Calmette-Guérin-Mediated Trained Immunity and Its Perspectives for Controlling Intracellular Infections.

The bacillus Calmette-Guérin (BCG) is an attenuated bacterium derived from virulent Mycobacterium bovis. It is the only licensed vaccine used for preventing severe forms of tuberculosis in children. Besides its specific effects against tuberculosis, BCG administration is also associated with beneficial non-specific effects (NSEs) following heterologous stimuli in humans and mice. The NSEs from BCG could be related to both adaptive and innate immune responses. The latter is also known as trained immunity (TI), a recently described biological feature of innate cells that enables functional improvement based on metabolic and epigenetic reprogramming. Currently, the mechanisms related to BCG-mediated TI are the focus of intense research, but many gaps are still in need of elucidation. This review discusses the present understanding of TI induced by BCG, exploring signaling pathways that are crucial to a trained phenotype in hematopoietic stem cells and monocytes/macrophages lineage. It focuses on BCG-mediated TI mechanisms, including the metabolic-epigenetic axis and the inflammasome pathway in these cells against intracellular pathogens. Moreover, this study explores the TI in different immune cell types, its ability to protect against various intracellular infections, and the integration of trained innate memory with adaptive memory to shape next-generation vaccines.

Bacillus Calmette-Guérin-Trained Macrophages Elicit a Protective Inflammatory Response against the Pathogenic Bacteria Brucella abortus.

The bacillus Calmette-Guérin (BCG) can elicit enhanced innate immune responses against a wide range of infections, known as trained immunity. Brucella abortus is the causative agent of brucellosis, a debilitating disease that affects humans and animals. In this study, we demonstrate that C57BL/6 mouse bone marrow-derived macrophages under BCG training enhance inflammatory responses against B. abortus. BCG-trained macrophages showed increased MHC class II and CD40 expression on the cell surface and higher IL-6, IL-12, and IL-1ß production. The increase in IL-1ß secretion was accompanied by enhanced activation of canonical and noncanonical inflammasome platforms. We observed elevated caspase-11 expression and caspase-1 processing in BCG-trained macrophages in response to B. abortus compared with untrained cells. In addition, these BCG-trained cells showed higher NLRP3 expression after B. abortus infection. From a metabolic point of view, signaling through the Akt/mammalian target of rapamycin/S6 kinase pathway was also enhanced. In addition, BCG training resulted in higher inducible NO synthase expression and nitrite production, culminating in an improved macrophage-killing capacity against intracellular B. abortus. In vivo, we monitored a significant reduction in the bacterial burden in organs from BCG-trained C57BL/6 mice when compared with the untrained group. In addition, previous BCG immunization of RAG-1-deficient mice partially protects against Brucella infection, suggesting the important role of the innate immune compartment in this scenario. Furthermore, naive recipient mice that received BM transfer from BCG-trained donors showed greater resistance to B. abortus when compared with their untrained counterparts. These results demonstrate that BCG-induced trained immunity in mice results in better control of intracellular B. abortus in vivo and in vitro.

Recombinant Bacillus Calmette-Guérin Expressing SARS-CoV-2 Chimeric Protein Protects K18-hACE2 Mice against Viral Challenge.

COVID-19 has accounted for more than 6 million deaths worldwide. Bacillus Calmette-Guérin (BCG), the existing tuberculosis vaccine, is known to induce heterologous effects over other infections due to trained immunity and has been proposed to be a potential strategy against SARS-CoV-2 infection. In this report, we constructed a recombinant BCG (rBCG) expressing domains of the SARS-CoV-2 nucleocapsid and spike proteins (termed rBCG-ChD6), recognized as major candidates for vaccine development. We investigated whether rBCG-ChD6 immunization followed by a boost with the recombinant nucleocapsid and spike chimera (rChimera), together with alum, provided protection against SARS-CoV-2 infection in K18-hACE2 mice. A single dose of rBCG-ChD6 boosted with rChimera associated with alum elicited the highest anti-Chimera total IgG and IgG2c Ab titers with neutralizing activity against SARS-CoV-2 Wuhan strain when compared with control groups. Importantly, following SARS-CoV-2 challenge, this vaccination regimen induced IFN-γ and IL-6 production in spleen cells and reduced viral load in the lungs. In addition, no viable virus was detected in mice immunized with rBCG-ChD6 boosted with rChimera, which was associated with decreased lung pathology when compared with BCG WT-rChimera/alum or rChimera/alum control groups. Overall, our study demonstrates the potential of a prime-boost immunization system based on an rBCG expressing a chimeric protein derived from SARS-CoV-2 to protect mice against viral challenge.

TLR9 activation is required for cytotoxic response elicited by baculovirus capsid display.

Although the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects lepidopteran invertebrates as natural hosts, represents an efficient vector for vaccine development. Baculovirus surface display induces strong humoral responses against viruses and parasites. A novel strategy based on capsid display carrying foreign antigens in the AcMNPV particle further improved the immune response by eliciting CD8+ T cell activation. In this study, we analyze the intracellular mechanisms and signalling pathways involved in CD8+ T cell activation by capsid display. Our results show that baculovirus can attach to the cell surface, enter dendritic cells (DCs), transit within endocytic vesicles and escape to the cytosol for further degradation by the proteasome. We found that the availability of viral proteins, endosomal acidification, and proteasome activity are needed for efficient Major Histocompatibility Complex class-I presentation by baculovirus carrying Ovalbumin in the viral capsid. Importantly, we demonstrated with this strategy that the induction of cytotoxic T cells and IL-12 production by DCs are TLR9-dependent and STING-independent. Finally, our study shows differential intracellular processing for capsid and surface baculovirus proteins in DCs and highlights the role of different danger receptors during cytotoxic T cell priming through the capsid display delivery system, which could lead to improved baculovirus-based vaccines development.

Recombinant bacillus calmette–guérin expressing SARS-CoV-2 chimeric protein protects K18-hACE2 mice against viral challenge

COVID-19 has accounted for more than 6 million deaths worldwide. Bacillus Calmette–Guérin (BCG), the existing tuberculosis vaccine, is known to induce heterologous effects over other infections due to trained immunity and has been proposed to be a potential strategy against SARS-CoV-2 infection. In this report, we constructed a recombinant BCG (rBCG) expressing domains of the SARS-CoV-2 nucleocapsid and spike proteins (termed rBCG-ChD6), recognized as major candidates for vaccine development. We investigated whether rBCG-ChD6 immunization followed by a boost with the recombinant nucleocapsid and spike chimera (rChimera), together with alum, provided protection against SARS-CoV-2 infection in K18-hACE2 mice. A single dose of rBCG-ChD6 boosted with rChimera associated with alum elicited the highest anti-Chimera total IgG and IgG2c Ab titers with neutralizing activity against SARS-CoV-2 Wuhan strain when compared with control groups. Importantly, following SARS-CoV-2 challenge, this vaccination regimen induced IFN-γ and IL-6 production in spleen cells and reduced viral load in the lungs. In addition, no viable virus was detected in mice immunized with rBCG-ChD6 boosted with rChimera, which was associated with decreased lung pathology when compared with BCG WT-rChimera/alum or rChimera/alum control groups. Overall, our study demonstrates the potential of a prime-boost immunization system based on an rBCG expressing a chimeric protein derived from SARS-CoV-2 to protect mice against viral challenge.

The endoplasmic reticulum stress sensor IRE1α modulates macrophage metabolic function during Brucella abortus infection.

Endoplasmic reticulum (ER) stress plays a major role in several inflammatory disorders. ER stress induces the unfolded protein response (UPR), a conserved response broadly associated with innate immunity and cell metabolic function in various scenarios. Brucella abortus, an intracellular pathogen, triggers the UPR via Stimulator of interferon genes (STING), an important regulator of macrophage metabolism during B. abortus infection. However, whether ER stress pathways underlie macrophage metabolic function during B. abortus infection remains to be elucidated. Here, we showed that the UPR sensor inositol-requiring enzyme 1α (IRE1α) is as an important component regulating macrophage immunometabolic function. In B. abortus infection, IRE1α supports the macrophage inflammatory profile, favoring M1-like macrophages. IRE1α drives the macrophage metabolic reprogramming in infected macrophages, contributing to the reduced oxidative phosphorylation and increased glycolysis. This metabolic reprogramming is probably associated with the IRE1α-dependent expression and stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), an important molecule involved in cell metabolism that sustains the inflammatory profile in B. abortus-infected macrophages. Accordingly, we demonstrated that IRE1α favors the generation of mitochondrial reactive oxygen species (mROS) which has been described as an HIF-1α stabilizing factor. Furthermore, in infected macrophages, IRE1α drives the production of nitric oxide and the release of IL-1ß. Collectively, these data unravel a key mechanism linking the UPR and the immunometabolic regulation of macrophages in Brucella infection and highlight IRE1α as a central pathway regulating macrophage metabolic function during infectious diseases.

MyD88-dependent BCG immunotherapy reduces tumor and regulates tumor microenvironment in bladder cancer murine model.

Bacillus Calmette-Guerin (BCG) is the only FDA approved first line therapy for patients with nonmuscle invasive bladder cancer. The purpose of this study is to better understand the role of innate immune pathways involved in BCG immunotherapy against murine bladder tumor. We first characterized the immunological profile induced by the MB49 mouse urothelial carcinoma cell line. MB49 cells were not able to activate an inflammatory response (TNF-α, IL-6, CXCL-10 or IFN-ß) after the stimulus with different agonists or BCG infection, unlike macrophages. Although MB49 cells are not able to induce an efficient immune response, BCG treatment could activate other cells in the tumor microenvironment (TME). We evaluated BCG intratumoral treatment in animals deficient for different innate immune molecules (STING-/-, cGAS-/-, TLR2-/-, TLR3-/-, TLR4-/-, TLR7-/-, TLR9-/-, TLR3/7/9-/-, MyD88-/-, IL-1R-/-, Caspase1/11-/-, Gasdermin-D-/- and IFNAR-/-) using the MB49 subcutaneous mouse model. Only MyD88-/- partially responded to BCG treatment compared to wild type (WT) mice, suggesting a role played by this adaptor molecule. Additionally, BCG intratumoral treatment regulates cellular infiltrate in TME with an increase of inflammatory macrophages, neutrophils and CD8+ T lymphocytes, suggesting an immune response activation that favors tumor remission in WT mice but not in MyD88-/-. The experiments using MB49 cells infected with BCG and co-cultured with macrophages also demonstrated that MyD88 is essential for an efficient immune response. Our data suggests that BCG immunotherapy depends partially on the MyD88-related innate immune pathway.

Galectin-3 regulates proinflammatory cytokine function and favours Brucella abortus chronic replication in macrophages and mice.

In this study, we provide evidence that galectin-3 (Gal-3) plays an important role in Brucella abortus infection. Our results showed increased Gal-3 expression and secretion in B. abortus infected macrophages and mice. Additionally, our findings indicate that Gal-3 is dispensable for Brucella-containing vacuoles disruption, inflammasome activation and pyroptosis. On the other hand, we observed that Brucella-induced Gal-3 expression is crucial for induction of molecules associated to type I IFN signalling pathway, such as IFN-ß: Interferon beta (IFN-ß), C-X-C motif chemokine ligand 10 (CXCL10) and guanylate-binding proteins. Gal-3 KO macrophages showed reduced bacterial numbers compared to wild-type cells, suggesting that Gal-3 facilitates bacterial replication in vitro. Moreover, priming Gal-3 KO cells with IFN-ß favoured B. abortus survival in macrophages. Additionally, we also observed that Gal-3 KO mice are more resistant to B. abortus infection and these animals showed elevated production of proinflammatory cytokines when compared to control mice. Finally, we observed an increased recruitment of macrophages, dendritic cells and neutrophils in spleens of Gal-3 KO mice compared to wild-type animals. In conclusion, this study demonstrated that Brucella-induced Gal-3 is detrimental to host and this molecule is implicated in inhibition of recruitment and activation of immune cells, which promotes B. abortus spread and aggravates the infection. TAKE AWAYS: Brucella abortus infection upregulates galectin-3 expression Galectin-3 regulates guanylate-binding proteins expression but is not required for Brucella-containing vacuole disruption Galectin-3 modulates proinflammatory cytokine production during bacterial infection Galectin-3 favours Brucella replication.

STING regulates metabolic reprogramming in macrophages via HIF-1α during Brucella infection.

Macrophages metabolic reprogramming in response to microbial insults is a major determinant of pathogen growth or containment. Here, we reveal a distinct mechanism by which stimulator of interferon genes (STING), a cytosolic sensor that regulates innate immune responses, contributes to an inflammatory M1-like macrophage profile upon Brucella abortus infection. This metabolic reprogramming is induced by STING-dependent stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), a global regulator of cellular metabolism and innate immune cell functions. HIF-1α stabilization reduces oxidative phosphorylation and increases glycolysis during infection with B. abortus and, likewise, enhances nitric oxide production, inflammasome activation and IL-1ß release in infected macrophages. Furthermore, the induction of this inflammatory profile participates in the control of bacterial replication since absence of HIF-1α renders mice more susceptible to B. abortus infection. Mechanistically, activation of STING by B. abortus infection drives the production of mitochondrial reactive oxygen species (mROS) that ultimately influences HIF-1α stabilization. Moreover, STING increases the intracellular succinate concentration in infected macrophages, and succinate pretreatment induces HIF-1α stabilization and IL-1ß release independently of its cognate receptor GPR91. Collectively, these data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during B. abortus infection that is orchestrated by STING via HIF-1α pathway and highlight the metabolic reprogramming of macrophages as a potential treatment strategy for bacterial infections.

Impact of STING Inflammatory Signaling during Intracellular Bacterial Infections.

The early detection of bacterial pathogens through immune sensors is an essential step in innate immunity. STING (Stimulator of Interferon Genes) has emerged as a key mediator of inflammation in the setting of infection by connecting pathogen cytosolic recognition with immune responses. STING detects bacteria by directly recognizing cyclic dinucleotides or indirectly by bacterial genomic DNA sensing through the cyclic GMP-AMP synthase (cGAS). Upon activation, STING triggers a plethora of powerful signaling pathways, including the production of type I interferons and proinflammatory cytokines. STING activation has also been associated with the induction of endoplasmic reticulum (ER) stress and the associated inflammatory responses. Recent reports indicate that STING-dependent pathways participate in the metabolic reprogramming of macrophages and contribute to the establishment and maintenance of a robust inflammatory profile. The induction of this inflammatory state is typically antimicrobial and related to pathogen clearance. However, depending on the infection, STING-mediated immune responses can be detrimental to the host, facilitating bacterial survival, indicating an intricate balance between immune signaling and inflammation during bacterial infections. In this paper, we review recent insights regarding the role of STING in inducing an inflammatory profile upon intracellular bacterial entry in host cells and discuss the impact of STING signaling on the outcome of infection. Unraveling the STING-mediated inflammatory responses can enable a better understanding of the pathogenesis of certain bacterial diseases and reveal the potential of new antimicrobial therapy.

Vaccines for COVID-19: perspectives from nucleic acid vaccines to BCG as delivery vector system.

This article discusses standard and new disruptive strategies in the race to develop an anti-COVID-19 vaccine. We also included new bioinformatic data from our group mapping immunodominant epitopes and structural analysis of the spike protein. Another innovative approach reviewed here is the use of BCG vaccine as priming strategy and/or delivery system expressing SARS-CoV-2 antigens.

Guanylate binding proteins contained in the murine chromosome 3 are important to control mycobacterial infection.

Guanylate binding proteins (GBPs) are important effector molecules of autonomous response induced by proinflammatory stimuli, mainly IFNs. The murine GBPs clustered in chromosome 3 (GBPchr3) contains the majority of human homologous GBPs. Despite intense efforts, mycobacterial-promoted diseases are still a major public health problem. However, the combined importance of GBPchr3 during mycobacterial infection has been overlooked. This study addresses the influence of the GBPchr3 in host immunity against mycobacterial infection to elucidate the relationship between cell-intrinsic immunity and triggering of an efficient anti-mycobacterial immune response. Here we show that all GBPchr3 are up-regulated in lungs of mice during Mycobacterium bovis BCG infection, resembling tissue expression of IFN-γ. Mice deficient in GBPchr3 (GBPchr3-/- ) were more susceptible to infection, displaying diminished expression of autophagy-related genes (LC3B, ULK1, and ATG5) in lungs. Additionally, there was reduced proinflammatory cytokine production complementary to diminished numbers of myeloid cells in spleens of GBPchr3-/- . Higher bacterial burden in GBPchr3-/- animals correlated with increased number of tissue granulomas. Furthermore, absence of GBPchr3 hampered activation and production of TNF-α and IL-12 by dendritic cells. Concerning macrophages, lack of GBPs impaired their antimicrobial function, diminishing autophagy induction and intracellular killing efficiency. In contrast, single GBP2 deficiency did not contribute to in vivo bacterial control. In conclusion, this study shows that GBPchr3 are important not only to stimulate cell-intrinsic immunity but also for inducing an efficient immune response to control mycobacterial infection in vivo.

The role of the adaptor molecule STING during Schistosoma mansoni infection.

Schistosomiasis is a human parasitic disease responsible for serious consequences for public health, as well as severe socioeconomic impacts in developing countries. Here, we provide evidence that the adaptor molecule STING plays an important role in Schistosoma mansoni infection. S. mansoni DNA is sensed by cGAS leading to STING activation in murine embryonic fibroblasts (MEFs). Sting-/- and C57BL/6 (WT) mice were infected with schistosome cercariae in order to assess parasite burden and liver pathology. Sting-/- mice showed worm burden reduction but no change in the number of eggs or granuloma numbers and area when compared to WT animals. Immunologically, a significant increase in IFN-γ production by the spleen cells was observed in Sting-/- animals. Surprisingly, Sting-/- mice presented an elevated percentage of neutrophils in lungs, bronchoalveolar lavage, and spleens. Moreover, Sting-/- neutrophils exhibited increased survival rate, but similar ability to kill schistosomula in vitro when stimulated with IFN-γ when compared to WT cells. Finally, microbiota composition was altered in Sting-/- mice, revealing a more inflammatory profile when compared to WT animals. In conclusion, this study demonstrates that STING signaling pathway is important for S. mansoni DNA sensing and the lack of this adaptor molecule leads to enhanced resistance to infection.

The Role of ST2 Receptor in the Regulation of Brucella abortus Oral Infection.

The ST2 receptor plays an important role in the gut such as permeability regulation, epithelium regeneration, and promoting intestinal immune modulation. Here, we studied the role of ST2 receptor in a murine model of oral infection with Brucella abortus, its influence on gut homeostasis and control of bacterial replication. Balb/c (wild-type, WT) and ST2 deficient mice (ST2-/-) were infected by oral gavage and the results were obtained at 3 and 14 days post infection (dpi). Our results suggest that ST2-/- are more resistant to B. abortus infection, as a lower bacterial colony-forming unit (CFU) was detected in the livers and spleens of knockout mice, when compared to WT. Additionally, we observed an increase in intestinal permeability in WT-infected mice, compared to ST2-/- animals. Breakage of the intestinal epithelial barrier and bacterial dissemination might be associated with the presence of the ST2 receptor; since, in the knockout mice no change in intestinal permeability was observed after infection. Together with enhanced resistance to infection, ST2-/- produced greater levels of IFN-γ and TNF-α in the small intestine, compared to WT mice. Nevertheless, in the systemic model of infection ST2 plays no role in controlling Brucella replication in vivo. Our results suggest that the ST2 receptor is involved in the invasion process of B. abortus by the mucosa in the oral infection model.

Vaccines for COVID-19: perspectives from nucleic acid vaccines to BCG as delivery vector system

This article discusses standard and new disruptive strategies in the race to develop an anti-COVID-19 vaccine. We also included new bioinformatic data from our group mapping immunodominant epitopes and structural analysis of the spike protein. Another innovative approach reviewed here is the use of BCG vaccine as priming strategy and/or delivery system expressing SARS-CoV-2 antigens.

Bacterial RNA Contributes to the Down-Modulation of MHC-II Expression on Monocytes/Macrophages Diminishing CD4+ T Cell Responses.

Brucella abortus, the causative agent of brucellosis, displays many resources to evade T cell responses conducive to persist inside the host. Our laboratory has previously showed that infection of human monocytes with B. abortus down-modulates the IFN-γ-induced MHC-II expression. Brucella outer membrane lipoproteins are structural components involved in this phenomenon. Moreover, IL-6 is the soluble factor that mediated MHC-II down-regulation. Yet, the MHC-II down-regulation exerted by lipoproteins was less marked than the one observed as consequence of infection. This led us to postulate that there should be other components associated with viable bacteria that may act together with lipoproteins in order to diminish MHC-II. Our group has recently demonstrated that B. abortus RNA (PAMP related to pathogens' viability or vita-PAMP) is involved in MHC-I down-regulation. Therefore, in this study we investigated if B. abortus RNA could be contributing to the down-regulation of MHC-II. This PAMP significantly down-modulated the IFN-γ-induced MHC-II surface expression on THP-1 cells as well as in primary human monocytes and murine bone marrow macrophages. The expression of other molecules up-regulated by IFN-γ (such as co-stimulatory molecules) was stimulated on monocytes treated with B. abortus RNA. This result shows that this PAMP does not alter all IFN-γ-induced molecules globally. We also showed that other bacterial and parasitic RNAs caused MHC-II surface expression down-modulation indicating that this phenomenon is not restricted to B. abortus. Moreover, completely degraded RNA was also able to reproduce the phenomenon. MHC-II down-regulation on monocytes treated with RNA and L-Omp19 (a prototypical lipoprotein of B. abortus) was more pronounced than in monocytes stimulated with both components separately. We also demonstrated that B. abortus RNA along with its lipoproteins decrease MHC-II surface expression predominantly by a mechanism of inhibition of MHC-II expression. Regarding the signaling pathway, we demonstrated that IL-6 is a soluble factor implicated in B. abortus RNA and lipoproteins-triggered MHC-II surface down-regulation. Finally, CD4+ T cells functionality was affected as macrophages treated with these components showed lower antigen presentation capacity. Therefore, B. abortus RNA and lipoproteins are two PAMPs that contribute to MHC-II down-regulation on monocytes/macrophages diminishing CD4+ T cell responses.

JVA, an isoniazid analogue, is a bioactive compound against a clinical isolate of the Mycobacterium avium complex.

Bacteria belonging to Mycobacterium avium complex are organisms of low pathogenicity that infect immunosuppressed individuals. Infection is treated with an antimicrobial macrolide, Clarithromycin (CAM) or Azitromycin, associated with Ethambutol and Rifabutin during 12 months. Regimen long duration and side effects hinder patient's commitment to treatment favoring emergence of antibiotic resistance. In this present study, we evaluated the activity of JVA, an Isoniazid (INH) derivative, against M. avium 2447, a clinical isolate. We demonstrated that JVA reduces M. avium 2447 growth in macrophages, more efficiently than CAM and INH. In order to explore JVA mechanism of action, we investigated compound properties and performed pH-dependent stability studies. Our results suggest an enhanced ability of JVA to cross biological membranes. Furthermore, we suggest that in acidic conditions of macrophages' phagosomes, where mycobacteria replicate, JVA would be promptly hydrolyzed to INH, delivering the adduct INH-nicotinamide adenine dinucleotide and thus inhibiting M. avium 2447 growth.

The cGAS/STING Pathway Is Important for Dendritic Cell Activation but Is Not Essential to Induce Protective Immunity against Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis (Mtb) infection remains a major public health concern. The STING (stimulator of interferon genes) pathway contributes to the cytosolic surveillance of host cells. Most studies on the role of STING activation in Mtb infection have focused on macrophages. Moreover, a detailed investigation of the role of STING during Mtb infection in vivo is required. Here, we deciphered the involvement of STING in the activation of dendritic cells (DCs) and the host response to Mtb infection in vivo. In DCs, this adaptor molecule was important for Ifn-ß expression and IL-12 production as well as for the surface expression of the activation markers CD40 and CD86. We also documented that Mtb DNA induces STING activation in murine fibroblasts. In vivo Mtb aerogenic infection induced the upregulation of the STING and cGAS (cyclic GMP-AMP synthase) genes, and Ifn-ß pulmonary expression was dependent on both sensors. However, mice deficient for STING or cGAS presented a similar outcome to wild-type controls, with no major alterations in body weight gain, bacterial burden, or survival. Lung inflammation, proinflammatory cytokine production, and inflammatory cell recruitment were similar in STING- and cGAS-deficient mice compared to wild-type controls. In summary, although the STING pathway seems to be crucial for DC activation during Mtb infection, it is dispensable for host protection in vivo.

The use of gold nanorods as a new vaccine platform against schistosomiasis.

Schistosomiasis is an important parasitic disease affecting >207 million people in 76 countries around the world and causing approximately 250,000 deaths per year. At present, the main strategy adopted for the control of schistosomiasis is the use of safe chemotherapy, such as praziquantel. However, the high rates of reinfection after treatment restrict the use of this treatment approach and assume the need for other forms of control such as vaccination. Sm29 is a protein that is localized in the Schistosoma mansoni tegument of adult worms and schistosomula and is considered a powerful vaccine candidate. Because of the chemical, physical and immunological characteristics of nanoparticles, nanocarriers have received increasing attention. In the field of nanotechnology, gold nanorods are considered potential vaccine carriers. In this study, we bound S. mansoni rSm29 protein to gold nanorods either directly or by cysteamine functionalization. When the worm burden was evaluated, the AuNRs-NH2-rSm29 group of immunized mice showed the best protection level (34%). Following AuNRs-NH2-rSm29 immunization, we observed a Th1 immunological response in mice with higher production of IFN-γ, mainly by CD4+ and CD8+ T cells. Furthermore, AuNRs-NH2-rSm29 could activate dendritic cells in vitro, enhancing MHCII and MHCI expression and the production of IL-1ß in a NLRP3-, ASC- and Caspase-1-dependent manner. In summary, our findings support the use of nanorods as an immunization strategy in vaccine development against infectious diseases.