Maria Cecilia Rodriguez-Galan

Toll-like receptors are key players in neurodegeneration

The activation of innate immune response is initiated by engagement of pattern-recognition receptors (PPRs), such as Toll-like receptors (TLRs). These receptors are expressed in peripheral leukocytes and in many cell types in the central nervous system (CNS). The expression of TLRs in CNS was mainly studied in astrocytes and microglial cells. However, new evidence indicates that these receptors may play an important role in neuronal homeostasis. The expression of TLRs in the CNS is variable and can be modulated by multiple factors, including pro-inflammatory molecules, which are elevated in neurodegenerative diseases and can increase the expression of TLRs in CNS cells. Moreover, activation of TLRs induces the release of pro-inflammatory cytokines. Therefore, TLRs have been shown to play a role in several aspects of neurodegenerative diseases. Here we will discuss results reported in the recent literature concerning the participation of TLRs in neurodegenerative diseases.

Autophagy in inflammation, infection, neurodegeneration and cancer.

In its classical form, autophagy is an essential, homeostatic process by which cytoplasmic components are degraded in a double-membrane-bound autophagosome in response to starvation. Paradoxically, although autophagy is primarily a protective process for the cell, it can also play a role in cell death. The roles of autophagy bridge both the innate and adaptive immune systems and autophagic dysfunction is associated with inflammation, infection, neurodegeneration and cancer. In this review, we discuss the contribution of autophagy to inflammatory, infectious and neurodegenerative diseases, as well as cancer.

Synergistic Effect of IL-2, IL-12, and IL-18 on Thymocyte Apoptosis and Th1/Th2 Cytokine Expression

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-γ in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-γ and IL-13. Analysis of the thymic subsets, CD4−CD8− (DN), CD4+CD8+, CD4+CD8−, and CD4−CD8+ revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-γ and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-γ+ cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-γ and IL-13 expression is dependent on Stat4 and NF-κB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-γ. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-γ-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.

Impaired Activity of Phagocytic Cells in Candida albicans Infection after Exposure to Chronic Varied Stress

Objective: Candidiasis is a prototypic opportunistic fungal disease that may follow severe modulations of the immune system of the host. The purpose of this study was to evaluate which innate immune mechanisms involved in the protection against fungal invasion are impaired under stress conditions. Methods: Wistar rats were infected intraperitoneally with Candida albicans and immediately exposed to chronic varied stress (CVS) over 10 days (CVS; Ca-S); the fungal burden (CFU), histopathological lesion and ACTH levels were evaluated. Additionally, functional assessment of peritoneal cells (PC) included the phagocytic and anticandidacidal activities and the production of H2O2 and NO. Results: In the only infected animals (Ca), C. albicans colonization stimulated an efficient inflammatory response, while in Ca-S rats poor tissue reactions were associated with increased CFU in livers and kidneys (p < 0.05, Ca vs. Ca-S). Whereas the phagocytic process was not modified, the candidacidal activity of PC was significantly decreased after the application of CVS (p < 0.001, Ca vs. Ca-S). The H2O2 production by macrophages and neutrophils was downregulated by the infection, and while at early intervals these cells possessed a residual oxidative capacity, by day 10, the production of this metabolite was blocked. Spontaneous NO production by macrophages was significantly increased in both Ca and Ca-S animals (p < 0.001), but in stressed rats, this reactive nitrogen intermediate was noticeably downregulated (p < 0.05, Ca vs. Ca-S). The hyperactivity of hypothalamus-pituitary-adrenal axis after exposure to stress was confirmed by an increase in baseline plasma ACTH levels. Conclusion: These results show that during infection with C. albicans, the exposure to CVS contributes to the spread of the fungus and downregulates critical functions of phagocytic cells involved in the control of this opportunistic pathogen.

MCP-1/CCR2 interactions direct migration of peripheral B and T lymphocytes to the thymus during acute infectious/inflammatory processes

Mature lymphocyte immigration into the thymus has been documented in mouse, rat, and pig models, and highly increases when cells acquire an activated phenotype. Entrance of peripheral B and T cells into the thymus has been described in healthy and pathological situations. However, it has not been proposed that leukocyte recirculation to the thymus could be a common feature occurring during the early phase of a Th1 inflammatory/infectious process when a large number of peripheral cells acquire an activated phenotype and the cellularity of the thymus is seriously compromised. The data we present here demonstrate that in well‐established Th1 models triggered by different types of immunogens, for example, LPS treatment (a bacterial product), Candida albicans infection (a fungus), and after Trypanosoma cruzi infection (a parasite), a large number of mature peripheral B and T cells enter the thymus. This effect is dependent on, but not exclusive of, the available space in the thymus. Our data also demonstrate that MCP‐1/CCR2 (where MCP‐1 is monocyte chemoattractant protein‐1) interaction is responsible for the infiltration of peripheral cells to the thymus in these Th1‐inflammatory/infectious situations. Finally, systemic expression of IL‐12 and IL‐18 produced during the inflammatory process is ultimately responsible for these migratory events.

Coexpression of IL-18 Strongly Attenuates IL-12-Induced Systemic Toxicity through a Rapid Induction of IL-10 without Affecting its Antitumor Capacity

IL-12 is an excellent candidate for the treatment of cancer due to its ability to drive strong antitumor responses. Recombinant IL-12 protein is currently used in cancer patients; however, systemic expression of rIL-12 presents disadvantages including cost and dose limitation due to its toxicity. In this study, we used hydrodynamic shear of cDNA as a tool to achieve systemic expression of IL-12. We found that sustained but toxic levels of serum IL-12 could be generated in 6- to 7-wk-old B6 mice after a single injection of the cDNA. Unexpectedly, we observed that when IL-12 cDNA is coinjected with IL-18 cDNA, IL-12 antitumor activity was maintained, but there was a significant attenuation of IL-12 toxicity, as evidenced by a greater survival index and a diminution of liver enzymes (ALT and AST). Interestingly, after IL-12 plus IL-18 cDNA administration, more rapid and higher IL-10 levels were observed than after IL-12 cDNA treatment alone. To understand the mechanism of protection, we coinjected IL-12 plus IL-10 cDNAs and observed an increase in survival that correlated with diminished serum levels of the inflammatory cytokines TNF-α and IFN-γ. Confirming the protective role of early IL-10 expression, we observed a significant decrease in survival in IL-10 knockout mice or IL-10R-blocked B6 mice after IL-12 plus IL-18 treatment. Thus, our data demonstrate that the high and early IL-10 expression induced after IL-12 plus IL-18 cDNA treatment is critical to rapidly attenuate IL-12 toxicity without affecting its antitumor capacity. These data could highly contribute to the design of more efficient/less toxic protocols for the treatment of cancer.

Hematopoiesis and Thymic Apoptosis Are Not Affected by the Loss of Cdk2

ABSTRACT Cell cycle regulation is essential for proper homeostasis of hematopoietic cells. Cdk2 is a major regulator of S phase entry, is activated by mitogenic cytokines, and has been suggested to be involved in antigen-induced apoptosis of T lymphocytes. The role of Cdk2 in hematopoietic cells and apoptosis in vivo has not yet been addressed. To determine whether Cdk2 plays a role in these cells, we performed multiple analyses of bone marrow cells, thymocytes, and splenocytes from Cdk2 knockout mice. We found that Cdk2 is not required in vivo to induce apoptosis in lymphocytes, a result that differs from previous pharmacological in vitro studies. Furthermore, thymocyte maturation was not affected by the lack of Cdk2. We then analyzed the hematopoietic stem cell compartment and found similar proportions of stem cells and progenitors in Cdk2−/− and wild-type animals. Knockouts of Cdk2 inhibitors (p21, p27) affect stem cell renewal, but a competitive graft experiment indicated that renewal and multilineage differentiation are normal in the absence of Cdk2. Finally, we stimulated T lymphocytes or macrophages to induce proliferation and observed normal reactivation of Cdk2−/− quiescent cells. Our results indicate that Cdk2 is not required for proliferation and differentiation of hematopoietic cells in vivo, although in vitro analyses consider Cdk2 to be a major player in proliferation and apoptosis in these cells and a potential target for therapy.

Interleukin 4 induces the apoptosis of mouse microglial cells by a caspase-dependent mechanism.

Microglial cells are resident macrophages in the central nervous system (CNS) and become activated in many pathological conditions. Activation of microglial cells results in reactive microgliosis, manifested by an increase in cell number in the affected CNS regions. The control of microgliosis may be important to prevent pathological damage to the brain. The type 2 cytokine IL-4 has been reported to be protective in brain inflammation. However, its effect on microglial cell survival was not well understood. In this study, we report a dual effect of IL-4 on the survival of mouse microglial cells. In a 6h short term culture, IL-4 reduced the death of microglial cells induced by staurosporine. In contrast, in long term treatment (more than 48h), IL-4 increased the apoptotic death of both primary mouse microglial cells and a microglial cell line N9. Mechanistic studies revealed that, in microglial cells, IL-4 increased the levels of cleaved caspase 3 and PARP, which is down-stream of activated caspase 3. In addition, IL-4 down regulated the autophagy and the antiapoptotic protein Bcl-xL in microglial cells. On the other hand, the pre-incubation of microglial cells with IL-4 for 24h, attenuated the cell death induced by the neurotoxic peptide amyloid beta 1-42 (Aβ42). Our observations demonstrate a novel function of IL-4 in regulating the survival of microglial cells, which may have important significance in reduction of undesired inflammatory responses in the CNS.

The Scaffold Protein Cybr Is Required for Cytokine-Modulated Trafficking of Leukocytes In Vivo

ABSTRACT Trafficking and cell adhesion are key properties of cells of the immune system. However, the molecular pathways that control these cellular behaviors are still poorly understood. Cybr is a scaffold protein highly expressed in the hematopoietic/immune system whose physiological role is still unknown. In vitro studies have shown it regulates LFA-1, a crucial molecule in lymphocyte attachment and migration. Cybr also binds cytohesin-1, a guanine nucleotide exchange factor for the ARF GTPases, which affects actin cytoskeleton remodeling during cell migration. Here we show that expression of Cybr in vivo is differentially modulated by type 1 cytokines during lymphocyte maturation. In mice, Cybr deficiency negatively affects leukocytes circulating in blood and lymphocytes present in the lymph nodes. Moreover, in a Th1-polarized mouse model, lymphocyte trafficking is impaired by loss of Cybr, and Cybr-deficient mice with aseptic peritonitis have fewer cells than controls present in the peritoneal cavity, as well as fewer leukocytes leaving the bloodstream. Mutant mice injected with Moloney murine sarcoma/leukemia virus develop significantly larger tumors than wild-type mice and have reduced lymph node enlargement, suggesting reduced cytotoxic T-lymphocyte migration. Taken together, these data support a role for Cybr in leukocyte trafficking, especially in response to proinflammatory cytokines in stress conditions.

Promoter Sequence of Shiga Toxin 2 (Stx2) Is Recognized In Vivo, Leading to Production of Biologically Active Stx2

ABSTRACT Shiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagic Escherichia coli (EHEC) strains. We previously described Stx2 expression by eukaryotic cells after they were transfected in vitro with the stx2 gene cloned into a prokaryotic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2 in vivo after pStx2 injection. Mice were inoculated by hydrodynamics-based transfection (HBT) with pStx2. We studied the survival, percentage of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNA was recovered from the liver, and Stx2 cytotoxic activity was observed in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, and markers of central nervous system (CNS) damage were observed, including increased expression of glial fibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressed in vivo from the wild stx2 gene, reproducing pathogenic damage induced by purified Stx2 or secondary to EHEC infection. IMPORTANCE Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC) infections are a serious public health problem, and Stx is the main pathogenic agent associated with typical hemolytic-uremic syndrome (HUS). In contrast to the detailed information describing the molecular basis for EHEC adherence to epithelial cells, very little is known about how Stx is released from bacteria in the gut, reaching its target tissues, mainly the kidney and central nervous system (CNS). In order to develop an efficient treatment for EHEC infections, it is necessary to understand the mechanisms involved in Stx expression. In this regard, the present study demonstrates that mammals can synthesize biologically active Stx using the natural promoter associated with the Stx-converting bacteriophage genome. These results could impact the comprehension of EHEC HUS, since local eukaryotic cells transduced and/or infected by bacteriophage encoding Stx2 could be an alternative source of Stx production. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC) infections are a serious public health problem, and Stx is the main pathogenic agent associated with typical hemolytic-uremic syndrome (HUS). In contrast to the detailed information describing the molecular basis for EHEC adherence to epithelial cells, very little is known about how Stx is released from bacteria in the gut, reaching its target tissues, mainly the kidney and central nervous system (CNS). In order to develop an efficient treatment for EHEC infections, it is necessary to understand the mechanisms involved in Stx expression. In this regard, the present study demonstrates that mammals can synthesize biologically active Stx using the natural promoter associated with the Stx-converting bacteriophage genome. These results could impact the comprehension of EHEC HUS, since local eukaryotic cells transduced and/or infected by bacteriophage encoding Stx2 could be an alternative source of Stx production.