Inhibition of hyperprogressive cancer disease induced by immune-checkpoint blockade upon co-treatment with meta-tyrosine and p38 pathway inhibitor.

BACKGROUND: Although immune-checkpoint inhibitors (ICI) are overall promissory for cancer treatment, they entail, in some cases, an undesired side-effect called hyperprogressive-cancer disease (HPD) associated with acceleration of tumor growth and shortened survival. METHODS: To understand the mechanisms of HPD we assayed the ICI therapy on two murine tumors widely different regarding immunogenicity and, subsequently, on models of local recurrences and metastases of these tumors. To potentiate the immune response (IR), we combined ICI with meta-tyrosine-that counteracts immune-suppressive signals-and a selective inhibitor of p38 pathway that proved to counteract the phenomenon of tumor-immunostimulation. RESULTS: ICI were therapeutically effective against both tumor models (proportionally to their immunogenicity) but only when they faced incipient tumors. In contrast, ICI produced acceleration of large and residual tumors. The combined treatment strongly inhibited the growth of large tumors and it managed to cure 80% of mice with local recurrences and 60% of mice bearing residual metastases. CONCLUSIONS: Tumor enhancement was paradoxically correlated to a weak increase of the antitumor IR suggesting that a weak IR - different from a strong tumor-inhibitory one-may produce stimulation of tumor growth, mimicking the HPD observed in some clinical settings.

Absence of interleukin-10 reduces progression of shiga toxin-induced hemolytic uremic syndrome.

Hemolytic Uremic Syndrome (HUS), a disease triggered by Shiga toxin (Stx), is characterized by hemolytic anemia, thrombocytopenia and renal failure. The inflammatory response mediated by polymorphonuclear neutrophils (PMNs) and monocytes is essential to HUS onset. Still, the role of anti-inflammatory cytokines is less clear. The deficiency of IL-10, an anti-inflammatory cytokine, leads to severe pathology in bacterial infections but also to beneficial effects in models of sterile injury. The aim of this work was to analyze the role of IL-10 during HUS. Control and IL-10 lacking mice (IL-10-/-) were intravenously injected with Stx type 2 (Stx2) and survival rate was evaluated. PMN and circulating and renal pro- and anti-inflammatory factors were analyzed by FACS and enzyme-linked immunosorbent assay (ELISA) respectively. IL-10-/- mice showed a higher survival associated with lower renal damage reflected by reduced plasma urea and creatinine levels than control mice. Circulating PMN increased at 72 h in both mouse strains accompanied by an up-regulation of CD11b in control mice. In parallel, renal PMN were significantly increased only in control mice after toxin. Plasma TNF-α, IL-6 and corticosterone levels were higher increased in IL-10-/- than control mice. Simultaneously renal TNF-α raised constantly but was accompanied by increased TGF-ß levels in IL-10-/- mice. These results demonstrate that the profile of circulating and renal cytokines after Stx2 differed between strains suggesting that balance of these factors could participate in renal protection. We conclude that IL-10 absence has a protective role in an experimental model of HUS by reducing PMN recruitment into kidney and renal damage, and increasing mice survival.

all-trans-retinoic acid improves immunocompetence in a murine model of lipopolysaccharide-induced immunosuppression.

Secondary infections due to post-sepsis immunosuppression are a major cause of death in patients with sepsis. Strategies aimed at restoring immune functions offer a new perspective in the treatment of sepsis. In the present study, we used LPS (lipopolysaccharide)-immunosuppressed mice to analyse the effects of ATRA (all-trans retinoic acid) on different immune parameters. The IS (immunocompromised) group had decreased lymphocyte and increased MDSC (myeloid-derived suppressor cell) counts in lymph nodes. They also had an impaired in vitro T-cell proliferation, mediated by MDSCs. ATRA administration restored T-cell proliferation, which was associated with a decreased number of live MDSCs. The IS group treated with ATRA had an increased number of CD4+ and CD8+ T-cells. ATRA partially improved the primary humoral immune response, even when immunosuppression was established first and ATRA was administered subsequently. Our results demonstrate that ATRA restores immunocompetence by modulating the number of leucocytes and the survival of MDSCs, and thus represents an additional potential strategy in the treatment of the immunosuppressive state of sepsis.

Interleukin-10 controls human peripheral PMN activation triggered by lipopolysaccharide.

Large amounts of anti-inflammatory mediators, such as interleukin (IL)-10, are produced and found early in the course of sepsis. We explore the role of IL-10 on neutrophil (PMN) activation/function using an in vitro model. Isolated human PMN were pre-incubated with lipopolysaccharide (LPS) and/or IL-10 for 18h. Subsequently, a second LPS exposure was performed and CD11b and CD66b up-regulation, and the reactive oxygen species (ROS) generation were measured 2h later. We found that IL-10 prevented PMN activation and the secretion of TNF-α and IL-8 induced by the first LPS contact. In the absence of IL-10, a second LPS exposure induced additive effects that were prevented by IL-10. Only ROS generation was highly affected by the blockade of PMN-secreted TNF-α or IL-8. Additionally, IL-10 prevented other possible mechanisms of LPS priming. Therefore, IL-10 modulates PMN activation preventing autocrine activating loops and priming mechanisms, rendering PMN less responsive to a second LPS exposure.

Peptidome profiling for the immunological stratification in sepsis: a proof of concept study.

Sepsis has been called the graveyard of pharmaceutical companies due to the numerous failed clinical trials. The lack of tools to monitor the immunological status in sepsis constrains the development of therapies. Here, we evaluated a test based on whole plasma peptidome acquired by MALDI-TOF-mass spectrometer and machine-learning algorithms to discriminate two lipopolysaccharide-(LPS) induced murine models emulating the pro- and anti-inflammatory/immunosuppression environments that can be found during sepsis. The LPS group was inoculated with a single high dose of LPS and the IS group was subjected to increasing doses of LPS, to induce proinflammatory and anti-inflammatory/immunosuppression profiles respectively. The LPS group showed leukopenia and higher levels of cytokines and tissue damage markers, and the IS group showed neutrophilia, lymphopenia and decreased humoral response. Principal component analysis of the plasma peptidomes formed discrete clusters that mostly coincided with the experimental groups. In addition, machine-learning algorithms discriminated the different experimental groups with a sensitivity of 95.7% and specificity of 90.9%. Data reveal the potential of plasma fingerprints analysis by MALDI-TOF-mass spectrometry as a simple, speedy and readily transferrable method for sepsis patient stratification that would contribute to therapeutic decision-making based on their immunological status.

Model of polymicrobial peritonitis that induces the proinflammatory and immunosuppressive phases of sepsis.

Severe sepsis is associated with early release of inflammatory mediators that contribute to the morbidity and mortality observed during the first stages of this syndrome. Although sepsis is a deadly, acute disease, high mortality rates have been observed in patients displaying evidence of sepsis-induced immune deactivation. Although the contribution of experimental models to the knowledge of pathophysiological and therapeutic aspects of human sepsis is undeniable, most of the current studies using animal models have focused on the acute, proinflammatory phase. We developed a murine model that reproduces the early acute phases but also the long-term consequences of human sepsis. We induced polymicrobial acute peritonitis (AP) by establishing a surgical connection between the cecum and the peritoneum, allowing the exit of intestinal bacteria. Using this model, we observed an acute phase with high mortality, leukopenia, increased interleukin-6 levels, bacteremia, and neutrophil activation. A peak of leukocytosis on day 9 or 10 revealed the persistence of the infection within the lung and liver, with inflammatory hepatic damage being shown by histological examination. Long-term (20 days) derangements in both innate and adaptive immune responses were found, as demonstrated by impaired systemic tumor necrosis factor alpha production in response to an inflammatory stimulus; a decreased primary humoral immune response and T cell proliferation, associated with an increased number of myeloid suppressor cells (Gr-1(+) CD11b(+)) in the spleen; and a low clearance capacity. This model provides a good approach to attempt novel therapeutic interventions directed to augmenting host immunity during late sepsis.

Shiga toxin 1-induced inflammatory response in lipopolysaccharide-sensitized astrocytes is mediated by endogenous tumor necrosis factor alpha.

Hemolytic-uremic syndrome (HUS) is generally caused by Shiga toxin (Stx)-producing Escherichia coli. Endothelial dysfunction mediated by Stx is a central aspect in HUS development. However, inflammatory mediators such as bacterial lipopolysaccharide (LPS) and polymorphonuclear neutrophils (PMN) contribute to HUS pathophysiology by potentiating Stx effects. Acute renal failure is the main feature of HUS, but in severe cases, patients can develop neurological complications, which are usually associated with death. Although the mechanisms of neurological damage remain uncertain, alterations of the blood-brain barrier associated with brain endothelial injury is clear. Astrocytes (ASTs) are the most abundant inflammatory cells of the brain that modulate the normal function of brain endothelium and neurons. The aim of this study was to evaluate the effects of Stx type 1 (Stx1) alone or in combination with LPS in ASTs. Although Stx1 induced a weak inflammatory response, pretreatment with LPS sensitized ASTs to Stx1-mediated effects. Moreover, LPS increased the level of expression of the Stx receptor and its internalization. An early inflammatory response, characterized by the release of tumor necrosis factor alpha (TNF-alpha) and nitric oxide and PMN-chemoattractant activity, was induced by Stx1 in LPS-sensitized ASTs, whereas activation, evidenced by higher levels of glial fibrillary acid protein and cell death, was induced later. Furthermore, increased adhesion and PMN-mediated cytotoxicity were observed after Stx1 treatment in LPS-sensitized ASTs. These effects were dependent on NF-kappaB activation or AST-derived TNF-alpha. Our results suggest that TNF-alpha is a pivotal effector molecule that amplifies Stx1 effects on LPS-sensitized ASTs, contributing to brain inflammation and leading to endothelial and neuronal injury.

Meta-tyrosine modulates the immune response induced by bacterial endotoxins.

Sepsis is characterized by an early pro-inflammatory phase followed by compensatory anti-inflammatory mechanisms that lead to a late generalized immunosuppression, period where most deaths occur. Immunotherapy approaches to recover the immunocompetence in sepsis are similar to those used in cancer. Meta-tyrosine (m-Tyr) is a product of oxidative stress present in circulation during the sepsis and cancer-associated pro-inflammatory stages. In this work, considering its potential participation in pro-inflammatory processes, we evaluate the effect of m-Tyr during LPS induced immunosuppression phase in a murine model. In addition, we examine the effect of m-Tyr in a vaccination strategy using a weakly immunogenic tumor model. Our results showed that m-Tyr could prevent the establishment of immunosuppression and rescue the host from an installed immunosuppression induced by LPS. These effects were parallel to the ability of m-Tyr to improve the pro-inflammatory effects induced by LPS and inhibit the anti-inflammatory action of dexamethasone. Also, m-Tyr treatment prevents both the reduction of splenic lymphocytes and the increase of the expression of programmed death ligand-1 in splenic myeloid cells associated with immunosuppression. Besides, treatment with m-Tyr increased the protective effect of an anti-tumor vaccine, suggesting that m-Tyr could improve the immune response. In summary, we suggest that m-Tyr can modulate critical immunological indicators through the inflammatory context, which could improve the management of diseases, such as sepsis and cancer, in which immunosuppression is a significant clinical problem.

Survival of an epidemic MDR strain of Mycobacterium tuberculosis and its non-prosperous variant within activated macrophages.

The fitness of a pathogen results from the interaction of multiple factors favoring either epidemiological success or failure. Herein, we studied the performance of the M strain, a highly successful multidrug resistant Mycobacterium tuberculosis genotype, and its non-prosperous variant, the 410 strain, in activated human monocyte-derived macrophages. Both strains showed comparable ability to induce necrotic cell death and to survive in apoptotic macrophages. Of the various macrophage activation conditions tested, none led to an enhanced control of the outbreak strain. The combination of 1,25(OH)2 vitaminD3 and IFN-γ favored significantly the control of the non-prosperous 410 strain. These observations indicate that the ability of the M strain to survive within the hostile intracellular milieu is conserved, and the overall fitness cost paid by this genotype would be low. Our results provide additional evidence on bacterial traits that may have contributed to the epidemiological success of the M strain.

Consequences of the Lack of IL-10 in Different Endotoxin Effects and its Relationship With Glucocorticoids.

Sepsis constitutes one of the major causes of death in ICUs. In sepsis induced by gram-negative, although lipopolysaccharide (LPS) initially induces an exacerbated secretion of proinflammatory cytokines leading to endotoxic shock and death resembling a septic shock, it is also capable of inducing refractoriness to subsequent challenge with LPS, a state known as endotoxin tolerance, which is considered the initial step of the immunosuppression found in septic patients. As we previously demonstrated the importance of glucocorticoids in endotoxin tolerance, the aim of this study was to evaluate the contribution of Interleukin-10 (IL-10) both in the endotoxic shock and in the development of the tolerance and its relationship with glucocorticoids. Our results show that, upon LPS challenge, IL-10 knockout mice (KO) mice had an enhanced LPS sensitivity, along with elevated levels of proinflammatory cytokines as tumor necrosis factor-α, IL-12 and interferon-γ, and enhanced tissue damage, despite the high levels of glucocorticoids. This effect may be because, in part, of the higher expression of tumor necrosis factor receptors in IL-10 KO mice. Further, the injection of dexamethasone did not protect IL-10 KO mice from a LPS lethal challenge. Although tolerance was achieved in the absence of IL-10, it was weaker and the elevated levels of glucocorticoids were not able to reverse the high sensitivity of IL-10 KO mice to LPS. Nevertheless, glucocorticoids would play a pivotal role in the establishment and maintenance of this partial tolerance in IL-10 KO mice. Finally, our results show that IL-10 and glucocorticoids could act in a bidirectional way influencing the inflammatory and anti-inflammatory periods.

Formation of Foamy Macrophages by Tuberculous Pleural Effusions Is Triggered by the Interleukin-10/Signal Transducer and Activator of Transcription 3 Axis through ACAT Upregulation.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its human host relies on numerous immune evasion strategies, such as the deregulation of the lipid metabolism leading to the formation of foamy macrophages (FM). Yet, the specific host factors leading to the foamy phenotype of Mtb-infected macrophages remain unknown. Herein, we aimed to address whether host cytokines contribute to FM formation in the context of Mtb infection. Our approach is based on the use of an acellular fraction of tuberculous pleural effusions (TB-PE) as a physiological source of local factors released during Mtb infection. We found that TB-PE induced FM differentiation as observed by the increase in lipid bodies, intracellular cholesterol, and expression of the scavenger receptor CD36, as well as the enzyme acyl CoA:cholesterol acyl transferase (ACAT). Importantly, interleukin-10 (IL-10) depletion from TB-PE prevented the augmentation of all these parameters. Moreover, we observed a positive correlation between the levels of IL-10 and the number of lipid-laden CD14+ cells among the pleural cells in TB patients, demonstrating that FM differentiation occurs within the pleural environment. Downstream of IL-10 signaling, we noticed that the transcription factor signal transducer and activator of transcription 3 was activated by TB-PE, and its chemical inhibition prevented the accumulation of lipid bodies and ACAT expression in macrophages. In terms of the host immune response, TB-PE-treated macrophages displayed immunosuppressive properties and bore higher bacillary loads. Finally, we confirmed our results using bone marrow-derived macrophage from IL-10-/- mice demonstrating that IL-10 deficiency partially prevented foamy phenotype induction after Mtb lipids exposure. In conclusion, our results evidence a role of IL-10 in promoting the differentiation of FM in the context of Mtb infection, contributing to our understanding of how alterations of the host metabolic factors may favor pathogen persistence.

[Reversion of the immunological eclipse and therapeutic vaccination against cancer in an experimental model]. / Reversion del eclipse inmunologico y vacunacion terapeutica contra el cancer en un modelo experimental.

Although animals can be prophylactically immunized against the growth of tumor implants, most of the attempts to use immunotherapy to cause the regression of animal and human tumors once they become established have been unsuccessful. To understand the nature of this refractoriness we have studied a methylcholanthrene-induced and strongly immunogenic murine fibrosarcoma. In our model, the onset of this refractoriness was associated with the beginning of an immunosuppressive state known as "immunological eclipse" characterized by a loss of the antitumor immune response when tumor grows beyond a critical size. This immunological eclipse was accompanied by the emergence of a systemic inflammatory condition. Treatment of tumor-bearing mice with a single dose of a synthetic corticosteroid, dexamethasone (DX), reduced significantly all parameters of systemic inflammation and simultaneously reversed the immunological eclipse. The reversion of the eclipse upon DX treatment was not curative itself, but allowed an immunological therapy based in dendritic cells pulsed with tumor antigens, which was itself absolutely ineffective, to exert a significant inhibitory effect against an established growing tumor. The two-step schedule using an anti-inflammatory treatment to reverse the immunological eclipse plus a dendritic cell-based vaccination strategy aimed to stimulate the antitumor immune response, could serve eventually as a model of immunotherapy against animal and human tumors.

Dehydroepiandrosterone and metyrapone partially restore the adaptive humoral and cellular immune response in endotoxin immunosuppressed mice.

Prior exposure to endotoxins renders the host temporarily refractory to subsequent endotoxin challenge (endotoxin tolerance). Clinically, this state has also been pointed out as the initial cause of the non-specific humoral and cellular immunosuppression described in these patients. We recently demonstrated the restoration of immune response with mifepristone (RU486), a receptor antagonist of glucocorticoids. Here we report the treatment with other modulators of glucocorticoids, i.e. dehydroepiandrosterone (DHEA), a hormone with anti-glucocorticoid properties, or metyrapone (MET) an inhibitor of corticosterone synthesis. These drugs were able to partially, but significantly, restore the humoral immune response in immunosuppressed mice. A significant recovery of proliferative responsiveness was also observed when splenocytes were obtained from DHEA- or MET-treated immunosuppressed mice. In addition, these treatments restored the hypersensitivity response in immunosuppressed mice. Finally, although neither DHEA nor MET improved the reduced CD4 lymphocyte count in spleen from immunosuppressed mice, both treatments promoted spleen architecture reorganization, partially restoring the distinct cellular components and their localization in the spleen. The results from this study indicate that DHEA and MET could play an important role in the restoration of both adaptive humoral and cellular immune response in LPS-immunosuppressed mice, reinforcing the concept of a central involvement of endogenous glucocorticoids on this phenomenon.

Reversión del eclipse inmunológico y vacunación terapéutica contra el cáncer en un modelo experimental / Reversion of the immunological eclipse and therapeutic vaccination against cancer in an experimental model

Aunque existen vacunas para prevenir la aparición de tumores en animales de experimentación, la mayoría de los intentos por aplicar aquellas vacunas con fines terapéuticos contra tumores establecidos no han sido exitosos. Para comprender la naturaleza de esta refractariedad, estudiamos un tumor de ratón fuertemente inmunogénico inducido por el carcinógeno químico metilcolantreno. En nuestro modelo, el inicio de esta refractariedad coincidió con el comienzo de un estado de inmunosupresión conocido como "eclipse inmunológico" caracterizado por una pérdida o bloqueo de la respuesta inmune antitumoral después que el tumor ha superado cierto tamaño crítico. Este eclipse inmunológico fue acompañado por un proceso de inflamación sistémica en el organismo. El tratamiento de los ratones portadores de tumor con una única dosis del corticoide sintético dexametasona (DX) redujo los parámetros de inflamación sistémica e indujo la reversión del eclipse. Esta reversión no fue por sí misma curativa pero permitió que un tratamiento inmunológico basado en células dendríticas estimuladas con antígenos tumorales, que por sí solo era absolutamente ineficaz, pudiera ejercer un significativo efecto inhibidor sobre un tumor en crecimiento. El esquema de dos pasos que comprende, primero, un tratamiento antiinflamatorio para revertir el eclipse y segundo, una estrategia de vacunación basada en células dendríticas destinada a estimular la respuesta inmune antitumoral, podría servir, eventualmente, como un modelo de inmunoterapia contra tumores en animales y seres humanos.

Although animals can be prophylactically immunized against the growth of tumor implants, most of the attempts to use immunotherapy to cause the regression of animal and human tumors once they become established have been unsuccessful. To understand the nature of this refractoriness we have studied a methylcholanthrene-induced and strongly immunogenic murine fibrosarcoma. In our model, the onset of this refractoriness was associated with the beginning of an immunosuppressive state known as "immunological eclipse" characterized by a loss of the antitumor immune response when tumor grows beyond a critical size. This immunological eclipse was accompanied by the emergence of a systemic inflammatory condition. Treatment of tumor-bearing mice with a single dose of a synthetic corticosteroid, dexamethasone (DX), reduced significantly all parameters of systemic inflammation and simultaneously reversed the immunological eclipse. The reversion of the eclipse upon DX treatment was not curative itself, but allowed an immunological therapy based in dendritic cells pulsed with tumor antigens, which was itself absolutely ineffective, to exert a significant inhibitory effect against an established growing tumor. The two-step schedule using an anti-inflammatory treatment to reverse the immunological eclipse plus a dendritic cell-based vaccination strategy aimed to stimulate the anti-tumor immune response, could serve eventually as a model of immunotherapy against animal and human tumors.