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2 Figuras, 1 Tabla, Microbiota have emerged as key modulators of both the carcinogenic process and the immune response against cancer cells, and, thus, it seems to influence the efficacy of immunotherapy. While most studies have focused on analyzing the influence of gut microbiota, its composition substantially differs from that in the lung. Here, we describe how microbial life in the lungs is associated with host immune status in the lungs and, thus, how the identification of the microbial populations in the lower respiratory tract rather than in the gut might be key to understanding the lung carcinogenic process and to predict the efficacy of different treatments. Understanding the influence of lung microbiota on host immunity may identify new therapeutic targets and help to design new immunotherapy approaches to treat lung cancer.

7 figures, 1 table., [Aims]: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis., [Methods]: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times., [Results]: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα., [Conclusions]: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology., This work was supported by grant SAF2017-83120-C2-1-R and SAF2014-54763-C2-2-R from the Ministry of Science, Innovation and Universities and FEDER (Group B29_17R, Aragon Government). MG and LS were supported by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. IUM was supported by a PhD fellowship from Aragon Government, MA was supported by a post-doctoral fellowship “Juan de la Cierva-formación” from the Ministry of Science, Innovation and Universities. JP was supported by ARAID Foundation.

6 Figures, [Background] Elimination of cancer cells by some stimuli like chemotherapy and radiotherapy activates anticancer immunity after the generation of damage‐associated molecular patterns, a process recently named immunogenic cell death (ICD). Despite the recent advances in cancer immunotherapy, very little is known about the immunological consequences of cell death activated by cytotoxic CD8+ T (Tc) cells on cancer cells, that is, if Tc cells induce ICD on cancer cells and the molecular mechanisms involved., [Methods] ICD induced by Tc cells on EL4 cells was analyzed in tumor by vaccinating mice with EL4 cells killed in vitro or in vivo by Ag-specific Tc cells. EL4 cells and mutants thereof overexpressing Bcl-XL or a dominant negative mutant of caspase-3 and wild-type mice, as well as mice depleted of Tc cells and mice deficient in perforin, TLR4 and BATF3 were used. Ex vivo cytotoxicity of spleen cells from immunized mice was analyzed by flow cytometry. Expression of ICD signals (calreticulin, HMGB1 and interleukin (IL)-1β) was analyzed by flow cytometry and ELISA., [Results] Mice immunized with EL4.gp33 cells killed in vitro or in vivo by gp33-specific Tc cells were protected from parental EL4 tumor development. This result was confirmed in vivo by using ovalbumin (OVA) as another surrogate antigen. Perforin and TLR4 and BATF3-dependent type 1 conventional dendritic cells (cDC1s) were required for protection against tumor development, indicating cross-priming of Tc cells against endogenous EL4 tumor antigens. Tc cells induced ICD signals in EL4 cells. Notably, ICD of EL4 cells was dependent on caspase-3 activity, with reduced antitumor immunity generated by caspase-3–deficient EL4 cells. In contrast, overexpression of Bcl-XL in EL4 cells had no effect on induction of Tc cell antitumor response and protection., [Conclusions] Elimination of tumor cells by Ag-specific Tc cells is immunogenic and protects against tumor development by generating new Tc cells against EL4 endogenous antigens. This finding helps to explain the enhanced efficacy of T cell-dependent immunotherapy and provide a molecular basis to explain the epitope spread phenomenon observed during vaccination and chimeric antigen receptor (CAR)-T cell therapy. In addition, they suggest that caspase-3 activity in the tumor may be used as a biomarker to predict cancer recurrence during T cell-dependent immunotherapies., The authors would like to acknowledge the use of Servicios Científico Técnicos del CIBA (IACS-Universidad de Zaragoza) and Servicios Apoyo Investigación de la Universidad de Zaragoza.

Cytotoxic CD8(+) T (Tc) cells are the main executors of transformed and cancer cells during cancer immunotherapy. The latest clinical results evidence a high efficacy of novel immunotherapy agents that modulate Tc cell activity against bad prognosis cancers. However, it has not been determined yet whether the efficacy of these treatments can be affected by selection of tumoural cells with mutations in the cell death machinery, known to promote drug resistance and cancer recurrence. Here, using a model of prophylactic tumour vaccination based on the LCMV-gp33 antigen and the mouse EL4 T lymphoma, we analysed the molecular mechanism employed by Tc cells to eliminate cancer cells in vivo and the impact of mutations in the apoptotic machinery on tumour development. First of all, we found that Tc cells, and perf and gzmB are required to efficiently eliminate EL4.gp33 cells after LCMV immunisation during short-term assays (1–4 h), and to prevent tumour development in the long term. Furthermore, we show that antigen-pulsed chemoresistant EL4 cells overexpressing Bcl-X(L) or a dominant negative form of caspase-3 are specifically eliminated from the peritoneum of infected animals, as fast as parental EL4 cells. Notably, antigen-specific Tc cells control the tumour growth of the mutated cells, as efficiently as in the case of parental cells. Altogether, expression of the anti-apoptotic mutations does not confer any advantage for tumour cells neither in the short-term survival nor in long-term tumour formation. Although the mechanism involved in the elimination of the apoptosis-resistant tumour cells is not completely elucidated, neither necroptosis nor pyroptosis seem to be involved. Our results provide the first experimental proof that chemoresistant cancer cells with mutations in the main cell death pathways are efficiently eliminated by Ag-specific Tc cells in vivo during immunotherapy and, thus, provide the molecular basis to treat chemoresistant cancer cells with CD8 Tc-based immunotherapy.

The aim of the present work was to compare the impact on the gut microbiota composition and in the systemic inflammation of two types of breads denominated as industrial and celta with different composition (wheat flour vs . complex flour and seeds) and fermentation process (2 h vs. 24 h). Faecal and blood samples of 10 mice were collected at: (1) - basal, (2) - after 21 days of industrial bread intake and (3) - after another 21 days of celta bread exposure. Significant differences in the gut microbiota composition were detected after the consumption of both breads, whereas the celta bread increased the concentration of Akkermansia and Mucispirillum , the industrial bread favour the Bacteroidetes proliferation and additionally systemic inflammation. In conclusion, the more consumed industrial processed bread might provoke systemic inflammation, probably in relation with its composition and manufacture process.

8 figures, 1 table.-- Electronic supplementary information available, If not properly regulated, the inflammatory immune response can promote carcinogenesis, as evident in colorectal cancer (CRC). Aiming to gain mechanistic insight into the link between inflammation and CRC, we perform transcriptomics analysis of human CRC, identifying a strong correlation between expression of the serine protease granzyme A (GzmA) and inflammation. In a dextran sodium sulfate and azoxymethane (DSS/AOM) mouse model, deficiency and pharmacological inhibition of extracellular GzmA both attenuate gut inflammation and prevent CRC development, including the initial steps of cell transformation and epithelial-to-mesenchymal transition. Mechanistically, extracellular GzmA induces NF-κB-dependent IL-6 production in macrophages, which in turn promotes STAT3 activation in cultured CRC cells. Accordingly, colon tissues from DSS/AOM-treated, GzmA-deficient animals present reduced levels of pSTAT3. By identifying GzmA as a proinflammatory protease that promotes CRC development, these findings provide information on mechanisms that link immune cell infiltration to cancer progression and present GzmA as a therapeutic target for CRC., This work was supported in part by FEDER/Gobierno de Aragón (group B29), Ministerio de Economia y Competitividad (SAF2014-54763-C2-1 and SAF2017-83120-C2-1-R to J.P. and SAF2014-54763-C2-2-R to E.M.G.), and Instituto de Salud Carlos III (PI13/00864 to L.M.-L.). Predoctoral grants/contracts from Fundacion Santander/Universidad de Zaragoza (to L.S. and M.A.A.), Gobierno de Aragon (to I.U.-M., L.C., L.S., and P.J.-S.), and FPU/Ministerio de Educación, Cultura y Deportes (to P.M.L.). I.U.-M. was supported by Fondo Garantía Empleo Juvenil/INAEM. M.A.A. has a Juan de la Cierva contract (Ministerio de Ciencia, Innovación y Universidades). J.P. was supported by Fundación Aragon I+D (ARAID).

Objective Granzyme A (GzmA) levels are elevated in the plasma and synovium of patients with rheumatoid arthritis (RA), suggesting involvement of this protease in the pathogenesis of the disease. GzmA contributes to sepsis by regulating the production of proinflammatory cytokines. The purpose of this study was to evaluate the contribution of GzmA to the pathogenesis of RA in vivo and to examine the possibility that GzmA acting via tumor necrosis factor (TNF) stimulates osteoclastogenesis. Methods Inflammatory arthritis induced by type II collagen was evaluated in wild-type, GzmA-deficient, and perforin-deficient mice. The osteoclastogenic potential of GzmA was examined in vitro using bone marrow cells and colony-forming unit–granulocyte–macrophage (CFU-GM) cells and in vivo using GzmA-deficient mice. Results Gene deletion of GzmA attenuated collagen-induced arthritis, including serum levels of proinflammatory cytokines, joint damage, and bone erosion in affected mice, suggesting that osteoclast activity is reduced in the absence of GzmA. Accordingly, GzmA-treated bone marrow cells produced multinucleated cells that fulfilled the criteria for mature osteoclasts: tartrate-resistant acid phosphatase (TRAP) activity, β integrin expression, calcitonin receptor expression, and resorptive activity on dentin slices. GzmA appeared to act without accessory cells, and its activity was not affected by osteoprotegerin, suggesting a minor contribution of RANKL. It also induced the expression and secretion of TNF. Neutralization of TNF or stimulation of CFU-GM cells from TNF–/– mice prevented GzmA-induced osteoclastogenesis. GzmA-deficient mice had reduced osteoclastogenesis in vivo (fewer calcitonin receptor–positive multinucleated cells and fewer transcripts for cathepsin K, matrix metalloproteinase 9, and TRAP in joints) and reduced serum levels of C-terminal telopeptide of type I collagen. Conclusion GzmA contributes to the joint destruction of RA partly by promoting osteoclast differentiation.

Colorectal cancer (CRC) is the third most common malignancy worldwide presenting high mortality due to low treatment efficacy. Existing evidence indicates that inflammatory bowel disease (IBD) is associated with a higher risk of developing CRC. Many murine models of inflammation-related colon carcinogenesis (CAC) have been developed to study colon carcinogenesis and novel treatments. A commonly used model involves the combination of a single dose of azoxymethane (AOM), together with three cycles of the inflammatory agent dextran sodium sulfate (DSS) (5 days in drinking water followed by a two-week rest). Following this protocol, around 50% of the animals develop CRCs after 45 days and almost 100% of animals after 60 days. During CAC development, immune cells, cytokines, and other immune mediators are involved in both tumorigenesis and the elimination of cancer cells during immunotherapy. Thus, the study of mucosal immune responses (including lamina propria mononuclear cells and intraepithelial lymphocytes) is important to understand the role of the immune system during development and therapy in CRC. Single immune cell suspensions from lamina propria and epithelium can be purified combining selective tissue digestion and Percoll gradient centrifugation. Isolated cells can be characterized using flow cytometry by analyzing surface antigens or intracellular cytokines and cytotoxic mediators or employed for further investigations like comparative studies of mRNA expression, cell-proliferation assay, protein analysis, or even functional cytotoxicity assays. The CAC model is useful to study the involvement of immune cells not only during the carcinogenesis process but, in addition, during the treatment with novel immunotherapy protocols.

1 Figura.- 1 Tabla, Pulmonary aspergillosis is a severe infectious disease caused by some members of the Aspergillus genus, that affects immunocompetent as well as immunocompromised patients. Among the different disease forms, Invasive Aspergillosis is the one causing the highest mortality, mainly, although not exclusively, affecting neutropenic patients. This genus is very well known by humans, since different sectors like pharmaceutical or food industry have taken advantage of the biological activity of some molecules synthetized by the fungus, known as secondary metabolites, including statins, antibiotics, fermentative compounds or colorants among others. However, during infection, in response to a hostile host environment, the fungal secondary metabolism is activated, producing different virulence factors to increase its survival chances. Some of these factors also contribute to fungal dissemination and invasion of adjacent and distant organs. Among the different secondary metabolites produced by Aspergillus spp. Gliotoxin (GT) is the best known and better characterized virulence factor. It is able to generate reactive oxygen species (ROS) due to the disulfide bridge present in its structure. It also presents immunosuppressive activity related with its ability to killmammalian cells and/or inactivate critical immune signaling pathways like NFkB. In this comprehensive review, we will briefly give an overview of the lung immune response against Aspergillus as a preface to analyse the effect of different secondary metabolites on the host immune response, with a special attention to GT. We will discuss the results reported in the literature on the context of the animal models employed to analyse the role of GT as virulence factor, which is expected to greatly depend on the immune status of the host: why should you hide when nobody is seeking for you? Finally, GT immunosuppressive activity will be related with different human diseases predisposing to invasive aspergillosis in order to have a global view on the potential of GT to be used as a target to treat IA., This work was supported by Fondo Social Europeo (FSE; Gobierno de Aragón), ASPANOA and by grants SAF2017-83120-C2-1-R, SAF2014- 54763-C2-1-R, SAF2014-54763-C2-2-R from Spanish Ministry of Economy and Competitivenes. MV-G was granted by a Río Hortega contract of National Institute of Health Carlos III (CM16/00236) and MA by a Juan de la Cierva contract of Spanish Ministry of Economy and Competitiveness (FJCI-2017-31629).

Serotonin (5-HT) is an essential gastrointestinal modulator whose effects regulate the intestinal physiology. 5-HT effects depend on extracellular 5-HT bioavailability, which is controlled by the serotonin transporter (SERT) expressed in both the apical and basolateral membranes of enterocytes. SERT is a critical target for regulating 5-HT levels and consequently, modulating the intestinal physiology. The deregulation of innate immune receptors has been extensively studied in inflammatory bowel diseases (IBD), where an exacerbated defense response to commensal microbiota is observed. Interestingly, many innate immune receptors seem to affect the serotonergic system, demonstrating a new way in which microbiota could modulate the intestinal physiology. Therefore, our aim was to analyze the effects of NOD1 activation on SERT function, as well as NOD1's interaction with other immune receptors such as TLR2 and TLR4. Our results showed that NOD1 activation inhibits SERT activity and expression in Caco-2/TC7 cells through the extracellular signal-regulated kinase (ERK) signaling pathway. A negative feedback between 5-HT and NOD1 expression was also described. The results showed that TLR2 and TLR4 activation seems to regulate NOD1 expression in Caco-2/TC7 cells. To assess the extend of cross-talk between NOD1 and TLRs, NOD1 expression was measured in the intestinal tract (ileum and colon) of wild type mice and mice with individual knockouts of TLR2, and TLR4 as well as double knockout TLR2/TLR4 mice. Hence, we demonstrate that NOD1 acts on the serotonergic system decreasing SERT activity and molecular expression. Additionally, NOD1 expression seems to be modulated by 5-HT and other immune receptors as TLR2 and TLR4. This study could clarify the relation between both the intestinal serotonergic system and innate immune system, and their implications in intestinal inflammation.

Background/Aims: Serotonin (5-HT) is a chief modulator of intestinal activity. The effects of 5-HT depend on its extracellular availability, which is mainly controlled by serotonin transporter (SERT), expressed in enterocytes. On the other hand, innate immunity, mediated by Toll-like receptors (TLRs) and nucleotide oligomerization domain (NOD)-like receptors (NLRs), is known to control intestinal microbiota and maintain intestinal homeostasis. The dysregulation of the intestinal serotonergic system and innate immunity has been observed in inflammatory bowel diseases (IBD), the incidence of which has severely increased all over the world. The aim of the present study, therefore, was to analyze the effect of NOD2 on intestinal SERT activity and expression, as well as to study the crosstalk of NOD2 with TLR2 and TLR4. Methods: Intestinal epithelial cell line Caco-2/TC7 was used to analyze SERT activity and SERT, NOD2, TLR2 and TLR4 molecular expression by real-time PCR and western blotting. Moreover, intestinal tract (ileum and colon) from mice deficient in TLR2, TLR4 or TLR2/4 receptors was used to test the interdependence of NOD2 with these TLR receptors. Results: NOD2 activation inhibits SERT activity in Caco-2/TC7 cells, mainly due to the decrement of SERT molecular expression, with RIP2/RICK being the intracellular pathway involved in this effect. This inhibitory effect on SERT would yield an increment of extracellular 5-HT availability. In this sense, 5-HT strongly inhibits NOD2 expression. In addition, NOD2 showed greater interdependence with TLR2 than with TLR4. Indeed, NOD2 expression significantly increased in both cells treated with TLR2 agonists and the intestinal tract of Tlr2-/- mice. Conclusions: It may be inferred from our data that NOD2 could play a role in intestinal pathophysiology not only through its inherent innate immune role but also due to its interaction with other receptors as TLR2 and the modulation of the intestinal serotonergic system decreasing SERT activity and expression.

Toll-like receptors (TLRs) recognise pathogen-derived molecules and play a critical role during the host innate and adaptive immune response. Brucella spp. are intracellular gram-negative bacteria including several virulent species, which cause a chronic zoonotic infection in a wide range of mammalian hosts known as brucellosis. Recently it was isolated from wild rodents a new Brucella species, Brucella microti, that was found to be pathogenic in mice. Using this species-specific model, it was previously found that CD8+ T cells are required to control this infection. In order to find out the role of TLR-mediated responses in the control of this pathogen, the course of infection of B. microti was analyzed over 3 weeks in wild-type (WT) and TLR knock out (KO) mice including TLR2-/-, TLR4-/-, TLR9-/-, TLR2x4-/- and TLR 2x4x9-/-. WT and single TLR2, TLR4 and TLR9 KO mice similarly control infection in liver and spleen. In contrast, bacterial clearance was delayed in TLR2x4-/- and TLR2x4x9-/- mice at 7 and 14 days post-infection. This defect correlated with impaired maturation and pro-inflammatory cytokine production in B. microti-infected dendritic cells from TLR2x4-/- and TLR2x4x9-/- mice. Finally, it was found that Tc cells from TLR2x4-/- and TLR2x4x9-/- mice showed reduced ability to inhibit growth of B. microti in macrophages, suggesting the involvement of TLR2 and 4 in the generation of specific Tc cells. Our findings indicate that TLR2 and TLR4 are required to control B. microti infection in mice and that this effect could be related to its participation in the maturation of dendritic cells and the generation of specific CD8+ Tc cells. □ Published

For more than 20 years perforin and granzymes (GZMs) have been recognized as key cell death executors of cytotoxic T (Tc) and natural killer (NK) cells during cancer immunosurveillance. In immune surveillance, perforin and GZMB, the most potent cytotoxic molecules, act mainly as antitumoral and anti-infectious factors. However, when expressed by immune regulatory cells they may contribute to immune evasion of specific cancer types. By contrast, the other major granzyme, GZMA, seems not to play a major role in Tc/NK cell-mediated cytotoxicity, but acts as a proinflammatory cytokine that might contribute to cancer development. Members of the GZM family also regulate other biological processes unrelated to cell death, such as angiogenesis, vascular integrity, extracellular matrix remodeling, and barrier function, all of which contribute to cancer initiation and progression. Thus, a new paradigm is emerging in the field of oncoimmunology. Can GZMs act as protumoral factors under some circumstances? We review the diverse roles of GZMs in cancer progression, and new therapeutic opportunities emerging from targeting these protumoral roles.

Brucellosis is a zoonosis of worldwide distribution with numerous animal host species. Since the novel isolation of Brucella spp. from marine mammals in 1994 the bacteria have been isolated from various marine mammal hosts. The marine mammal reference strains Brucella pinnipedialis 12890 (harbour seal, Phoca vitulina) and Brucella ceti 12891 (harbour porpoise, Phocoena phocoena) were included in genus Brucella in 2007, however, their pathogenicity in the mouse model is pending. Herein this is evaluated in BALB/c mice with Brucella suis 1330 as a control. Both marine mammal strains were attenuated, however, B. ceti was present at higher levels than B. pinnipedialis in blood, spleen and liver throughout the infection, in addition B. suis and B. ceti were isolated from brains and faeces at times with high levels of bacteraemia. In B. suis-infected mice serum cytokines peaked at day 7. In B. pinnipedialis-infected mice, levels were similar, but peaked predominantly at day 3 and an earlier peak in spleen weight likewise implied an earlier response. The inflammatory response induced pathology in the spleen and liver. In B. ceti-infected mice, most serum cytokine levels were comparable to those in uninfected mice, consistent with a limited inflammatory response, which also was indicated by restricted spleen and liver pathology. Specific immune responses against all three strains were detected in vitro after stimulation of splenocytes from infected mice with the homologous heat-killed brucellae. Antibody responses in vivo were also induced by the three brucellae. The immunological pattern of B. ceti in combination with persistence in organs and limited pathology has heretofore not been described for other brucellae. These two marine mammal wildtype strains show an attenuated pattern in BALB/c mice only previously described for Brucella neotomea. This work was supported by the grants RTA 2013-00065-C02-01 from The National Institute for Agricultural and Food Research and Technology (INIA) and agrifood research in Spain (MPJdB) and SAF2014-54763-C2 from the European Social Fund (JP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Published