Systemic sterile induced-co-expression of IL-12 and IL-18 drive IFN-γ-dependent activation of microglia and recruitment of MHC-II-expressing inflammatory monocytes into the brain.

The development of neuroinflammation, as well as the progression of several neurodegenerative diseases, has been associated with the activation and mobilization of the peripheral immune system due to systemic inflammation. However, the mechanism by which this occurs remains unclear. Here, we addressed the effect of systemic sterile induced-co-expression of IL-12 and IL-18, in the establishment of a novel cytokine-mediated model of neuroinflammation. Following peripheral hydrodynamic shear of IL-12 plus IL-18 cDNAs in C57BL/6 mice, we induced systemic and persistent level of IL-12, which in turn promoted the elevation of circulating pro-inflammatory cytokines TNF-α and IFN-γ, accompanied with splenomegaly. Moreover, even though we identified an increased gene expression of both TNF-α and IFN-γ in the brain, we observed that only IFN-γ, but not TNF-α signaling through its type I receptor, was required to induce both the trafficking of leukocytes from the periphery toward the brain and upregulate MHC-II in microglia and inflammatory monocytes. Therefore, only TNF-α was shown to be dispensable, revealing an IFN-γ-dependent activation of microglia and recruitment of leukocytes, particularly of highly activated inflammatory monocytes. Taken together, our results argue for a systemic cytokine-mediated establishment and development of neuroinflammation, having identified IFN-γ as a potential target for immunomodulation.

Safety levels of systemic IL-12 induced by cDNA expression as a cancer therapeutic.

Aim: The aim of this work is to utilize a gene expression procedure to safely express systemic IL-12 and evaluate its effects in mouse tumor models. Materials & methods: Secondary lymphoid organs and tumors from EL4 and B16 tumor-bearing mice were analyzed by supervised and unsupervised methods. Results: IL-12 cDNA induced systemic IL-12 protein levels lower than the tolerated dose in patients. Control of tumor growth was observed in subcutaneous B16 and EL4 tumors. Systemic IL-12 expression induced a higher frequency of both total tumor-infiltrated CD45+ cells and proliferative IFN-γ+CD8+ T cells along with a lower frequency of CD4+FOXP3+ and CD11b+Gr-1+ cells. Conclusion: This approach characterizes the systemic effects of IL-12, helping to improve treatment of metastases or solid tumors.

Lay abstract IL-12 has emerged as a potent cytokine in mediating antitumor activity in preclinical models of cancer. However, this antitumor response has not yet been translated into the clinic because of toxic side effects. The aim of our work is to analyze the effects of IL-12 in mouse tumor models. We demonstrate that one injection of IL-12 cDNA can induce systemic IL-12 levels in serum even lower than the tolerated dose in patients. At this dose, an efficient control of tumor growth can be observed. We found a higher frequency of both total tumor-infiltrated leukocytes and IFN-γ-producing CD8⁺ T cells along with a lower frequency of regulatory CD4⁺FOXP3⁺ and CD11b⁺Gr1⁺ cells. Our work demonstrates that IL-12 cDNA can safely be used to treat cancer.

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.

Promoter sequence of Shiga toxin 2 (Stx2) is recognized in vivo, leading to production of biologically active Stx2.

UNLABELLED: 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.

Toll-like receptor 2 ligands promote microglial cell death by inducing autophagy.

Microglial cells are phagocytes in the central nervous system (CNS) and become activated in pathological conditions, resulting in microgliosis, manifested by increased cell numbers and inflammation in the affected regions. Thus, controlling microgliosis is important to prevent pathological damage to the brain. Here, we evaluated the contribution of Toll-like receptor 2 (TLR2) to microglial survival. We observed that activation of microglial cells with peptidoglycan (PGN) from Staphylococcus aureus and other TLR2 ligands results in cell activation followed by the induction of autophagy and autophagy-dependent cell death. In C57BL/6J mice, intracerebral injection of PGN increased the autophagy of microglial cells and reduced the microglial/macrophage cell number in brain parenchyma. Our results demonstrate a novel role of TLRs in the regulation of microglial cell activation and survival, which are important for the control of microgliosis and associated inflammatory responses in the CNS.

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 (Aß42). 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.

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.

Immune-metabolic balance in stressed rats during Candida albicans infection.

We evaluated the host metabolic response to chronic varied stress during infection with the fungus Candida albicans. We used four groups of female Wistar rats: normal uninfected and unstressed, stressed, C. albicans infected and infected, and stressed. Infected rats reacted with rapid metabolic adjustments, evident as anorexia and body weight loss, partly mediated by glucocorticoids and TNF-alpha. Higher circulating levels of IL-6 and glucose (p < 0.05) revealed the progress and catabolic effect of the inflammatory response. Infected and stressed rats instead showed anorexia associated with infection and weight loss as the result of reduced food intake. This group exhibited a prompt reduction in circulating leptin on day 3 (p < 0.05), reduction in glucose levels and depletion of hepatic glycogen depots. We also evaluated the contribution of TNF-alpha, glucocorticoids, and food deprivation to liver damage. Lipid peroxidation in liver detected in the infected and infected-stressed groups was exacerbated by the glucocorticoid receptor antagonist RU 486, suggesting the modulatory activity of glucocorticoids, while hepatic fat accumulation and glycogen depletion decreased with anti-TNF-alpha treatment. Food deprivation exacerbated liver injury while the response to stress contributed to greater fungal colonization. Our findings emphasize the impact of metabolic alterations on tissue damage when the host immune activity is modulated by stress mediators.

Analysis of murine natural killer cell microsomal proteins using two-dimensional liquid chromatography coupled to tandem electrospray ionization mass spectrometry.

This study describes the application of a single tube sample preparation technique coupled with multidimensional fractionation for the analysis of a complex membrane protein sample from murine natural killer (NK) cells. A solution-based method that facilitates the solubilization and tryptic digestion of integral membrane proteins is conjoined with strong cation exchange (SCX) liquid chromatography (LC) fractionation followed by microcapillary reversed-phase (microRP) LC tandem mass spectrometric analysis of each SCXLC fraction in second dimension. Sonication in buffered methanol solution was employed to solubilize, and tryptically digest murine NK cell microsomal proteins, allowing for the large-scale identification of integral membrane proteins, including the mapping of the membrane-spanning peptides. Bioinformatic analysis of the acquired tandem mass spectra versus the murine genome database resulted in 11,967 matching tryptic peptide sequences, corresponding to 5782 unique peptide identifications. These peptides resulted in identification of 2563 proteins of which 876 (34%) are classified as membrane proteins.