Validation of the Tetracycline Regulatable Gene Expression System for the Study of the Pathogenesis of Infectious Disease

Understanding the pathogenesis of infectious disease requires the examination and successful integration of parameters related to both microbial virulence and host responses. As a practical and powerful method to control microbial gene expression, including in vivo, the tetracycline-regulatable system has recently gained the favor of many investigative groups. However, some immunomodulatory effects of the tetracyclines, including doxycycline, could potentially limit its use to evaluate host responses during infection. Here we have used a well-established murine model of disseminated candidiasis, which is highly dependent on both the virulence displayed by the fungal cells and on the host immune status, to validate the use of this system. We demonstrate that the pathogenesis of the wild type C. albicans CAF2-1 strain, which does not contain any tet-regulatable element, is not affected by the presence of doxycycline. Moreover levels of key cytokines, chemokines and many other biomarkers, as determined by multi-analyte profiling, remain essentially unaltered by the presence of the antibiotic during infection. Our results indicate that the levels of doxycycline needed to control the tetracycline regulatable promoter gene expression system have no detectable effect on global host responses during candidiasis. Because tet-regulatable systems are now being increasingly used in a variety of pathogenic microorganisms, these observations have wide implications in the field of infectious diseases

Acute stress reduces wound-induced activation of microbicidal potential of ex vivo isolated human monocyte-derived macrophages

BACKGROUND: Psychological stress delays wound healing but the precise underlying mechanisms are unclear. Macrophages play an important role in wound healing, in particular by killing microbes. We hypothesized that (a) acute psychological stress reduces wound-induced activation of microbicidal potential of human monocyte-derived macrophages (HMDM), and (b) that these reductions are modulated by stress hormone release. METHODS: Fourty-one healthy men (mean age 35±13 years) were randomly assigned to either a stress or stress-control group. While the stress group underwent a standardized short-term psychological stress task after catheter-induced wound infliction, stress-controls did not. Catheter insertion was controlled. Assessing the microbicidal potential, we investigated PMA-activated superoxide anion production by HMDM immediately before and 1, 10 and 60 min after stress/rest. Moreover, plasma norepinephrine and epinephrine and salivary cortisol were repeatedly measured. In subsequent in vitro studies, whole blood was incubated with norepinephrine in the presence or absence of phentolamine (norepinephrine blocker) before assessing HMDM microbicidal potential. RESULTS: Compared with stress-controls, HMDM of the stressed subjects displayed decreased superoxide anion-responses after stress (p's <.05). Higher plasma norepinephrine levels statistically mediated lower amounts of superoxide anion-responses (indirect effect 95% CI: 4.14-44.72). Norepinephrine-treated HMDM showed reduced superoxide anion-production (p<.001). This effect was blocked by prior incubation with phentolamine. CONCLUSIONS: Our results suggest that acute psychological stress reduces wound-induced activation of microbicidal potential of HMDM and that this reduction is mediated by norepinephrine. This might have implications for stress-induced impairment in wound healing

Molecules Altering the Intracellular Thiol Content Modulate NF-kB and STAT-1/IRF-1 Signalling Pathways and IL-12 p40 and IL-27 p28 Production in Murine Macrophages

Background: The aim of this study was to investigate the molecular mechanisms involved in the production of Th1 cytokines, namely IL-12 and IL-27, when the intra-macrophage redox state was altered by different chemical entities such as GSH-C4, which is reduced glutathione carrying an aliphatic chain, or I-152, a pro-drug of N-acetyl-cysteine (NAC) and betamercaptoethylamine. We had already demonstrated that GSH-C4 and I-152 could shift the immune response towards Th1 in Ovalbumin-immunized mice as well as enhance Th1 response in HIV-1 Tat-immunized mice. Methodology/Principal Findings: By a new high performance liquid chromatography method, we found that 20 mM GSHC4 provided a number of thiol species in the form of GSH, while 20 mM I-152 decreased GSH and increased the thiols in the form of NAC and I-152. Under these experimental conditions, GSH-C4 and I-152 enhanced and suppressed respectively the mRNA expression levels of IL-12 p40 induced by LPS/IFN-c as assessed by Real-Time PCR. The protein production of IL-12 p40 was increased by GSH-C4 and decreased by I-152 as determined by Enzyme-linked immunosorbent assay. Western immunoblot and electrophoretic mobility shift assays revealed that Nuclear Factor -kB (NF-kB) activation was inhibited by I- 152 and prolonged by GSH-C4. Twenty mM I-152 stimulated IL-27 p28 gene expression and sustained Signal Transducer and Activator of Transcription (STAT)-mediated interferon regulator factor 1 (IRF-1) de novo synthesis. By contrast, 20 mM GSHC4 did not exert any effect on IL-27 p28 gene expression. Conclusions and Significance: an increase in the intra-macrophage redox state by GSH-C4 and I-152 enhances Th1 cytokine production although the chemical structure and the intra-cellular metabolism influence differently signalling pathways involved in IL-27 or IL-12 production. GSH-C4 and I-152 may be used as Th1 immunomodulators in some pathologies and in ageing where GSH depletion may contribute to the Th1/Th2 imbalance, and in new immunization strategies

Aging affects the responsiveness of rat peritoneal macrophages to GM-CSF and IL-4

Macrophages undergo significant functional alterations during aging. The aim of the present study was to investigate changes of rat macrophage functions and response to M1/M2 polarization signals with age. Therefore, resident and thioglycollate-elicited peritoneal macrophages from young (3-month-old) and aged (18-19-month-old) rats were tested for phagocytic capacity and ability to secrete inflammatory mediators following in vitro stimulation with LPS and GM-CSF, and IL-4, prototypic stimulators for classically (M1) and alternatively activated (M2) macrophages, respectively. Aging increased the frequency of monocyte-derived (CCR7+ CD68+) and the most mature (CD163+ CD68+) macrophages within resident and thioglycollate-elicited peritoneal macrophages, respectively. The ability to phagocyte zymosan of none of these two cell subsets was affected by either LPS and GM-CSF or IL-4. The upregulated production of IL-1 beta, IL-6 and IL-10 and downregulated that of TGF-beta was observed in response to LPS in resident and thioglycollate-elicited macrophages from rats of both ages. GM-CSF elevated production of IL-1 beta and IL-6 in resident macrophages from aged rats and in thioglycollate-elicited macrophages from young rats. Unexpectedly, IL-4 augmented production of proinflammatory mediators, IL-1 beta and IL-6, in resident macrophages from aged rats. In both resident and thioglycollate-elicited macrophages aging decreased NO/urea ratio, whereas LPS but not GM-SCF, shifted this ratio toward NO in the macrophages from animals of both ages. Conversely, IL-4 reduced NO/urea ratio in resident and thioglycollate-elicited macrophages from young rats only. In conclusion, our study showed that aging diminished GM-CSF-triggered polarization of elicited macrophages and caused paradoxical IL-4-driven polarization of resident macrophages toward proinflammatory M1 phenotype. This age-related deregulation of macrophage inflammatory mediator secretion and phagocytosis in response to M1/M2 activators may lead to the deficient control of infectious and/or inflammatory diseases in advanced age