Bacillus Calmette-Guérin immunotherapy induces an efficient antitumor response to control murine melanoma depending on MyD88 signaling.

Bacillus Calmette-Guérin (BCG) is the first line treatment for bladder cancer and it is also proposed for melanoma immunotherapy. BCG modulates the tumor microenvironment (TME) inducing an antitumor effective response, but the immune mechanisms involved still poorly understood. The immune profile of B16-F10 murine melanoma cells was assessed by infecting these cells with BCG or stimulating them with agonists for different innate immune pathways such as TLRs, inflammasome, cGAS-STING and type I IFN. B16-F10 did not respond to any of those stimuli, except for type I IFN agonists, contrasting with bone marrow-derived macrophages (BMDMs) that showed high production of proinflammatory cytokines. Additionally, we confirmed that BCG is able to infect B16-F10, which in turn can activate macrophages and spleen cells from mice in co-culture experiments. Furthermore, we established a subcutaneous B16-F10 melanoma model for intratumoral BCG treatment and compared wild type mice to TLR2-/-, TLR3-/-, TLR4-/-, TLR7-/-, TLR3/7/9-/-, caspase 1-/-, caspase 11-/-, IL-1R-/-, cGAS-/-, STING-/-, IFNAR-/-, MyD88-/-deficient animals. These results in vivo demonstrate that MyD88 signaling is important for BCG immunotherapy to control melanoma in mice. Also, BCG fails to induce cytokine production in the co-culture experiments using B16-F10 and BMDMs or spleen cells derived from MyD88-/- compared to wild-type (WT) animals. Immunotherapy with BCG was not able to induce the recruitment of inflammatory cells in the TME from MyD88-/- mice, impairing tumor control and IFN-γ production by T cells. In conclusion, MyD88 impacts on both innate and adaptive responses to BCG leading to an efficient antitumor response against melanoma.

Dual neutrophil subsets exacerbate or suppress inflammation in tuberculosis via IL-1ß or PD-L1.

Neutrophils can be beneficial or deleterious during tuberculosis (TB). Based on the expression of MHC-II and programmed death ligand 1 (PD-L1), we distinguished two functionally and transcriptionally distinct neutrophil subsets in the lungs of mice infected with mycobacteria. Inflammatory [MHC-II-, PD-L1lo] neutrophils produced inflammasome-dependent IL-1ß in the lungs in response to virulent mycobacteria and "accelerated" deleterious inflammation, which was highly exacerbated in IFN-γR-/- mice. Regulatory [MHC-II+, PD-L1hi] neutrophils "brake" inflammation by suppressing T-cell proliferation and IFN-γ production. Such beneficial regulation, which depends on PD-L1, is controlled by IFN-γR signaling in neutrophils. The hypervirulent HN878 strain from the Beijing genotype curbed PD-L1 expression by regulatory neutrophils, abolishing the braking function and driving deleterious hyperinflammation in the lungs. These findings add a layer of complexity to the roles played by neutrophils in TB and may explain the reactivation of this disease observed in cancer patients treated with anti-PD-L1.

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.

Leishmania braziliensis exosomes activate human macrophages to produce proinflammatory mediators.

Exosomes, organelles measuring 30-200nm, are secreted by various cell types. Leishmania exosomes consist of many proteins, including heat shock proteins, annexins, Glycoprotein 63, proteins exerting signaling activity and those containing mRNA and miRNA. Studies have demonstrated that Leishmania donovani exosomes downregulate IFN-γ and inhibit the expression of microbicidal molecules, such as TNF and nitric oxide, thus creating a microenvironment favoring parasite proliferation. Despite lacking immunological memory, data in the literature suggest that, following initial stimulation, mononuclear phagocytes may become "trained" to respond more effectively to subsequent stimuli. Here we characterized the effects of macrophage sensitization using L. braziliensis exosomes prior to infection by the same pathogen. Human macrophages were stimulated with L. braziliensis exosomes and then infected with L. braziliensis. Higher levels of IL-1ß and IL-6 were detected in cultures sensitized prior to infection compared to unstimulated infected cells. Moreover, stimulation with L. braziliensis exosomes induced macrophage production of IL-1ß, IL-6, IL-10 and TNF. Inhibition of exosome secretion by L. braziliensis prior to macrophage infection reduced cytokine production and produced lower infection rates than untreated infected cells. Exosome stimulation also induced the consumption/regulation of NLRP3 inflammasome components in macrophages, while the blockade of NLRP3 resulted in lower levels of IL-6 and IL-1ß. Our results suggest that L. braziliensis exosomes stimulate macrophages, leading to an exacerbated inflammatory state that may be NLRP3-dependent.

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.

Gasdermin-D activation promotes NLRP3 activation and host resistance to Leishmania infection.

Intracellular parasites from the Leishmania genus cause Leishmaniasis, a disease affecting millions of people worldwide. NLRP3 inflammasome is key for disease outcome, but the molecular mechanisms upstream of the inflammasome activation are still unclear. Here, we demonstrate that despite the absence of pyroptosis, Gasdermin-D (GSDMD) is active at the early stages of Leishmania infection in macrophages, allowing transient cell permeabilization, potassium efflux, and NLRP3 inflammasome activation. Further, GSDMD is processed into a non-canonical 25 kDa fragment. Gsdmd-/- macrophages and mice exhibit less NLRP3 inflammasome activation and are highly susceptible to infection by several Leishmania species, confirming the role of GSDMD for inflammasome-mediated host resistance. Active NLRP3 inflammasome and GSDMD are present in skin biopsies of patients, demonstrating activation of this pathway in human leishmaniasis. Altogether, our findings reveal that Leishmania subverts the normal functions of GSDMD, an important molecule to promote inflammasome activation and immunity in Leishmaniasis.

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.

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.

Practice-Changing Use of the 21-Gene Test for the Management of Patients With Early-Stage Breast Cancer in Latin America.

PURPOSE: We present a physician survey of the impact of 21-gene Breast Recurrence Score test results on treatment decisions in clinical practice in Latin America. METHODS: This prospective survey enrolled consecutive patients at 14 sites in Argentina, Colombia, Mexico, and Peru who had routine 21-gene testing. Physician surveys captured patient and tumor characteristics and treatment decisions before and after 21-gene test results. The survey spanned the period before and after Trial Assigning Individualized Options for Treatment (TAILORx) results reported (June 2018). Overall net percent change in adjuvant chemotherapy recommendations was estimated, and asymptotic 95% CIs with continuity correction were calculated. The proportion with a change between pretest treatment recommendation and actual treatment received was calculated overall and by Recurrence Score groups per TAILORx. RESULTS: Between March 2015 and December 2019, the survey was completed for 647 patients; 20% were node-positive. The mean patient age was 54 years (24-85 years); 55% were postmenopausal; 17%, 63%, and 20% had grade 1, 2, and 3 tumors, respectively; and 30% had tumors > 2 cm. Recurrence Score (RS) results were as follows: 20% RS 0-10, 56% RS 11-25, and 24% RS 26-100. Overall, chemotherapy recommendations fell by a relative proportion of 39% (95% CI, 33.4 to 44.3) after 21-gene testing (33% decrease in node-negative and 55% decrease in node-positive). Among node-negative patients, the relative decrease in chemotherapy recommendations was 28% (95% CI, 18.9 to 39.5) before TAILORx and 36% (95% CI, 28.4 to 43.7) after. CONCLUSION: To our knowledge, this large survey of 21-gene test practice patterns was the first conducted in Latin America and showed the relevance of 21-gene testing in low- and medium-resource countries to minimize chemotherapy overuse and underuse in breast cancer. The results showed substantial reductions in chemotherapy use overall-especially after TAILORx reported-indicating the practice-changing potential of that study.

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.

Substantial Reduction in Adjuvant Chemotherapy With the Use of the 21-Gene Test to Manage Early Breast Cancer in a Public Hospital in Brazil.

PURPOSE: We evaluated the impact of 21-gene test results on treatment decisions for patients with early-stage breast cancer treated under the public health care system in Brazil, Sistema Único de Saúde. METHODS: Eligible patients treated at Hospital Pérola Byington and Santa Casa de Misericórdia de São Paulo in Brazil were required to have the following characteristics: postsurgery with hormone receptor-positive, human epidermal growth factor 2-negative, node-negative and node-positive, and T1/T2 breast cancer and patients with these characteristics were candidates for adjuvant systemic therapy. Treatment recommendations, chemotherapy plus hormonal therapy (CT + HT) or HT alone, were captured before and after 21-gene test results. RESULTS: From August 2018 to April 2019, 179 women were enrolled. The mean age was 58 years (29-86 years), 135 (76%) were postmenopausal, and 58 (32%) had node-positive breast cancer. Most patients (61%) had a tumor > 2 cm, including 7% with tumors > 4 cm. Using Recurrence Score (RS) result cut points on the basis of the TAILORx trial, 40 (22%) had RS 0-10, 91 (51%) had RS 11-25, and 48 (27%) had RS 26-100. Before 21-gene testing, 162 of 179 (91%) patients were recommended for CT. After testing, 117 of 179 patients (65%) had changes in CT recommendation: 112 (63%) who were initially recommended CT received HT alone and five (3%) who were initially recommended HT alone received CT + HT. After 21-gene testing, 99% of physicians reported strong confidence in their treatment recommendations. CONCLUSION: The change in clinical practice at these public hospitals was greater than expected: 66% of initial treatment recommendations were changed to omit CT with 21-gene test results. Clinicopathologic features did not correlate well with 21-gene test results and did not adequately identify those most likely to benefit from CT.

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.

Lack of Interleukin-6 Affects IFN-γ and TNF-α Production and Early In Vivo Control of Brucella abortus Infection.

Interleukin-6 (IL-6) is a pleiotropic cytokine promptly produced in response to infections, which contributes to host defense through the stimulation of acute phase immune responses. Brucella abortus is an intracellular bacterium that causes chronic disease in humans and domestic animals and triggers a robust immune response, characterized by the production of inflammatory cytokines. However, the mechanisms of IL-6-related immune responses in the context of Brucella infections are not completely understood. In this report, we describe an increased susceptibility of IL-6 knockout (KO) mice in the early phase of Brucella infection. Furthermore, we demonstrate that IL-6 is required for interferon (IFN)-γ and tumor necrosis factor (TNF)-α induction by infected splenocytes, indicating a protective role for IL-6 against B. abortus that parallels with Th1 type of immune response. Additionally, IL-6 KO mice exhibited reduced splenomegaly during the early phase of the infection. Corroborating this result, IL-6 KO mice displayed reduced numbers of macrophages, dendritic cells, and neutrophils in the spleen and reduced myeloperoxidase activity in the liver compared to wild-type infected mice. However, we demonstrate that IL-6 is not involved in B. abortus intracellular restriction in mouse macrophages. Taken together, our findings demonstrate that IL-6 contributes to host resistance during the early phase of B. abortus infection in vivo, and suggest that its protective role maybe partially mediated by proinflammatory immune responses and immune cell recruitment.

Canonical and Non-canonical Inflammasome Activation by Outer Membrane Vesicles Derived From Bordetella pertussis.

Outer Membrane Vesicles (OMVs) derived from different Gram-negative bacteria have been proposed as an attractive vaccine platform because of their own immunogenic adjuvant properties. Pertussis or whooping cough is a highly contagious vaccine-preventable respiratory disease that resurged during the last decades in many countries. In response to the epidemiological situation, new boosters have been incorporated into vaccination schedules worldwide and new vaccine candidates have started to be designed. Particularly, our group designed a new pertussis vaccine candidate based on OMVs derived from Bordetella pertussis (BpOMVs). To continue with the characterization of the immune response induced by our OMV based vaccine candidate, this work aimed to investigate the ability of OMVs to activate the inflammasome pathway in macrophages. We observed that NLRP3, caspase-1/11, and gasdermin-D (GSDMD) are involved in inflammasome activation by BpOMVs. Moreover, we demonstrated that BpOMVs as well as transfected B. pertussis lipooligosaccharide (BpLOS) induce caspase-11 (Casp11) and guanylate-binding proteins (GBPs) dependent non-canonical inflammasome activation. Our results elucidate the mechanism by which BpOMVs trigger one central pathway of the innate response activation that is expected to skew the adaptive immune response elicited by BpOMVs vaccination.

Schistosoma antigens as activators of inflammasome pathway: from an unexpected stimulus to an intriguing role.

Parasites of the genus Schistosoma are organisms capable of living for decades within the definitive host. They interfere with the immune response by interacting with host's receptors. In this review, we discuss from the first reports to the most recent discoveries regarding the ability of Schistosoma antigens in triggering intracellular receptors and inducing inflammasome activation.

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.

Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case.

The serum amyloid A (SAA) gene family is highly conserved and encodes acute phase proteins that are upregulated in response to inflammatory triggers. Over the years, a considerable amount of literature has been published attributing a wide range of biological effects to SAAs such as leukocyte recruitment, cytokine and chemokine expression and induction of matrix metalloproteinases. Furthermore, SAAs have also been linked to protumorigenic, proatherogenic and anti-inflammatory effects. Here, we investigated the biological effects conveyed by murine SAA3 (mu rSAA3) recombinantly expressed in Escherichia coli. We observed the upregulation of a number of chemokines including CCL2, CCL3, CXCL1, CXCL2, CXCL6 or CXCL8 following stimulation of monocytic, fibroblastoid and peritoneal cells with mu rSAA3. Furthermore, this SAA variant displayed potent in vivo recruitment of neutrophils through the activation of TLR4. However, a major problem associated with proteins derived from recombinant expression in bacteria is potential contamination with various bacterial products, such as lipopolysaccharide, lipoproteins and formylated peptides. This is of particular relevance in the case of SAA as there currently exists a discrepancy in biological activity between SAA derived from recombinant expression and that of an endogenous source, i.e. inflammatory plasma. Therefore, we subjected commercial recombinant mu rSAA3 to purification to homogeneity via reversed-phase high-performance liquid chromatography (RP-HPLC) and re-assessed its biological potential. RP-HPLC-purified mu rSAA3 did not induce chemokines and lacked in vivo neutrophil chemotactic activity, but retained the capacity to synergize with CXCL8 in the activation of neutrophils. In conclusion, experimental results obtained when using proteins recombinantly expressed in bacteria should always be interpreted with care.

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.