Differential Expression of Inflammatory Cytokines and Stress Genes in Male and Female Mice in Response to a Lipopolysaccharide Challenge.

Sex plays a key role in an individual's immune response against pathogenic challenges such that females fare better when infected with certain pathogens. It is thought that sex hormones impact gene expression in immune cells and lead to sexually dimorphic responses to pathogens. We predicted that, in the presence of E. coli gram-negative lipopolysaccharide (LPS), there would be a sexually dimorphic response in proinflammatory cytokine production and acute phase stress gene expression and that these responses might vary among different mouse strains and times in a pattern opposite to that of body temperature associated with LPS-induced shock.Interleukin-6 (IL-6), macrophage inflammatory protein-Iβ (MIP-1β), tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) as well as beta-fibrinogen (Fgb) and metallothionein-1 (Mt-1) mRNA expression were measured at four time points (0, 2, 4 and 7 hours) after injection of E. coli LPS in mice from three inbred strains.Statistical analysis using analyses of variance (ANOVAs) showed that the levels of the all six traits changed over time, generally peaking at 2 hours after LPS injection. Mt-1, Fgb, and IL-6 showed differences among strains, although these were time-specific. Sexual dimorphism was seen for Fgb and IL6, and was most pronounced at the latest time period (7 hours) where male levels exceeded those for females. Trends for all six cytokine/gene expression traits were negatively correlated with those for body temperatures.The higher levels of expression of Fgb and IL6 in males compared with females are consistent with the greater vulnerability of males to infection and subsequent inflammation. Temperature appears to be a useful proxy for mortality in endotoxic shock, but sexual dimorphism in cytokine and stress gene expression levels may persist after an LPS challenge even if temperatures in the two sexes are similar and have begun to stabilize

Effect of sex and strain on the expression of IL-6.

<p>Expression of serum IL-6, a proinflammatory cytokine, tested using quantitative ELISAs. (Mean ± SEM) IL-6 data was transformed by raising all values to 0.3. At 0 hours, a significant difference was observed, with any difference in sex or strain being dependent on the other variable. At 7 hours, a significant difference was observed, with any difference in sex or strain being dependent on the other variable. Differing letters indicate significance. P < 0.05.</p

Beta-fibrinogen expression over time in three strains.

<p>Expression of Fgb mRNA as compared to control Actb mRNA following amplification of both genes with quantitative real time PCR. (Mean ± SEM) Fgb data was transformed by raising all values to 0.2. A. There was a significant strain by time interaction (<i>F</i> = 5.55; d.f. = 6, 91; <i>P</i> < 0.0001), with post-hoc tests showing differences in expression levels between BALB/c and B6 mice at 7 hours, and between BL6 and both other strains at 4 hours B. There was also a sex by time interaction (<i>F</i> = 6.73; d.f. = 3, 91; <i>P</i> = 0.0004)), with higher expression levels in males compared with females at the 2 hour and especially the 7 hour time periods, but the reverse at the 4 hour time period. Means with different superscripts are significantly different. P < 0.05.</p

Time effect of cytokine expression.

<p>Concentration of serum cytokines observed from quantitative ELISAs. Due to transformations of all data, graphs are shown with arbitrary units. (Mean ± SEM) A. A significant difference was observed in the expression of MIP-1β, a chemokine that attracts macrophages, over the three time points tested. MIP-1β data was transformed by raising all values to 0.4. B. A significant difference was observed in expression of IL-1β, a proinflammatory cytokine, in the three time points tested. IL-1B, unlike others tested, did not show as high variation over the three time points. IL-1β data was transformed by raising all values to 0.25. C. A significant difference was observed in the expression of TNF-α, a proinflammatory cytokine, over the three time points tested. TNF-α data was transformed by raising all values to 0.5. Means with different superscripts within cytokine bars are significantly different. P < 0.05.</p

Body temperature changes over time following LPS exposure.

<p>Following a non-lethal dosage of 5mg/kg of LPS, mice were tested over a 9-hour time period for changes in body temperature. (Mean ± SEM) These were plotted by sex and strain. A. A significant difference in temperature was observed between male and female BALB/c mice at 3 hours and at 4 hours following LPS injection. B. A significant difference in temperature was observed between male and female CD1 mice at 2 hours, but not at any other time points measured. C. A significant difference was observed between male and female C57BL/6 mice at 4 and 5 hours following LPS injection. *P < 0.05.</p

Effect of strain on the expression of metallothionein-1 in mice.

<p>Expression of Mt-1, a stress gene expressed in the liver, as compared to the control gene, Actb, following amplification with quantitative real time PCR. (Mean ± SEM) Mt-1 data was transformed by raising all values to 0.4. A. At 0 hours, a significant difference between BALB/c mice and C57BL/6 mice was observed. Neither was significantly different from CD1 mice in terms of Mt-1 expression. B. At 4 hours, a significant difference was observed from baseline levels for all strains. At this time, the levels were slightly higher for all three strains than the levels observed at 0 hours. F = 3.06; d.f. = 6,00; *P = 0.0 Means with different superscripts within time points are significantly different.</p

Human microglia and astrocytes constitutively express the neurokinin-1 receptor and functionally respond to substance P

Abstract Background The tachykinin substance P (SP) is recognized to exacerbate inflammation at peripheral sites via its target receptor, neurokinin 1 receptor (NK-1R), expressed by leukocytes. More recently, SP/NK-1R interactions have been associated with severe neuroinflammation and neuronal damage. We have previously demonstrated that NK-1R antagonists can limit neuroinflammatory damage in a mouse model of bacterial meningitis. Furthermore, we have since shown that these agents can attenuate bacteria-induced neuronal and glial inflammatory mediator production in nonhuman primate (NHP) brain explants and isolated neuronal cells, and following in vivo infection. Methods In the present study, we have assessed the ability of NHP brain explants, primary human microglia and astrocytes, and immortalized human glial cell lines to express NK-1R isoforms. We have utilized RT-PCR, immunoblot analysis, immunofluorescent microscopy, and/or flow cytometric analysis, to quantify NK-1R expression in each, at rest, or following bacterial challenge. Furthermore, we have assessed the ability of human microglia to respond to SP by immunoblot analysis of NF-kB nuclear translocation and determined the ability of this neuropeptide to augment inflammatory cytokine release and neurotoxic mediator production by human astrocytes using an ELISA and a neuronal cell toxicity assay, respectively. Results We demonstrate that human microglial and astrocytic cells as well as NHP brain tissue constitutively express robust levels of the full-length NK-1R isoform. In addition, we demonstrate that the expression of NK-1R by human astrocytes can be further elevated following exposure to disparate bacterial pathogens or their components. Importantly, we have demonstrated that NK-1R is functional in both human microglia and astrocytes and show that SP can augment the inflammatory and/or neurotoxic immune responses of glial cells to disparate and clinically relevant bacterial pathogens. Conclusions The robust constitutive and functional expression of the full-length NK-1R isoform by human microglia and astrocytes, and the ability of SP to augment inflammatory signaling pathways and mediator production by these cells, support the contention that SP/NK-1R interactions play a significant role in the damaging neuroinflammation associated with conditions such as bacterial meningitis

Vesiculovirus Neutralization by Natural IgM and Complement

Because of its very low human seroprevalence, vesicular stomatitis virus (VSV) has promise as a systemic oncolytic agent for human cancer therapy. However, as demonstrated in this report, the VSV infectious titer drops by 4 log units during the first hour of exposure to nonimmune human serum. This neutralization occurs relatively slowly and is mediated by the concerted actions of natural IgM and complement. Maraba virus, whose G protein is about 80% homologous to that of VSV, is relatively resistant to the neutralizing activity of nonimmune human serum. We therefore constructed and rescued a recombinant VSV whose G gene was replaced by the corresponding gene from Maraba virus. Comparison of the parental VSV and VSV with Maraba G substituted revealed nearly identical host range properties and replication kinetics on a panel of tumor cell lines. Moreover, in contrast to the parental VSV, the VSV with Maraba G substituted was resistant to nonimmune human serum. Overall, our data suggest that VSV with Maraba G substituted should be further investigated as a candidate for human systemic oncolytic virotherapy applications. IMPORTANCE Oncolytic virotherapy is a promising approach for the treatment of disseminated cancers, but antibody neutralization of circulating oncolytic virus particles remains a formidable barrier. In this work, we developed a pseudotyped vesicular stomatitis virus (VSV) with a glycoprotein of Maraba virus, a closely related but serologically distinct member of the family Rhabdoviridae, which demonstrated greatly diminished susceptibility to both nonimmune and VSV-immune serum neutralization. VSV with Maraba G substituted or lentiviral vectors should therefore be further investigated as candidates for human systemic oncolytic virotherapy and gene therapy applications