Endotoxin tolerance (ET) is a phenomenon in which previous exposure of cells or organisms to microbial products, such as the endotoxin, LPS, induces a transient period of hypo-responsiveness to a subsequent endotoxin challenge. Exposure to an endotoxin initiates the production of pro-inflammatory cytokines and a subsequent production of anti-inflammatory cytokines by immune cells [7
]. ET is characterized by diminished release of pro-inflammatory cytokines, such as IL-1β, IL-1α, and TNF-α, and increased expression of anti-inflammatory cytokines, such as 1L-10 [14
]. This negative feedback mechanism is important for protecting the host from tissue damage and death caused by excessive inflammation The differential expression of cytokines/chemokines in different tissues and at different times has been examined in Sprague Dawley rats to investigate the modulation of immune responses [46
The occurrence of ET has been shown to increase the incidence of several diseases, including sepsis, pancreatitis, trauma and surgery [55
]. The CDC has identified the increase of HIV-1 positive population as a major factor in the increased incidence of sepsis [44
]. Studies have shown that the presence of HIV viral proteins causes a compromised immune response in HIV-1 patients [56
]. The phenomenon of ET during HIV-1 infection has not been studied through animal models. Reid and colleagues [32
] established a non-infectious HIV-1 transgenic (HIV-1Tg) rat model that expresses an HIV-1 provirus regulated by the viral promoter, but with a functional deletion of gag
. The characteristics of the HIV-1Tg rat include immunologic dysfunction, nephropathy, muscle wasting, skin lesions, and cataracts. We studied the systemic effect of the concurrent presence HIV-1 viral proteins and ET on the inflammatory response to bacterial endotoxin using the HIV-1Tg rat model. We examined the LPS-induced gene expression of 84 cytokines, chemokines, and their receptors in the blood, brain, and spleen of the endotoxin tolerant HIV-1Tg rat. Based on previous studies, we used two injections of a low dose of LPS (250 μg/kg) administered 9 h apart to induce ET, and a challenge injection with a high dose of LPS (5 mg/kg) 12 h later. A single exposure to a high dose of endotoxin should cause a significant increase in the levels of pro-inflammatory and anti-inflammatory cytokines compared to control [46
]. In the ET state, when animals are exposed to repeated low doses of endotoxin, upon a subsequent challenge with a high dose of the endotoxin, one would expect that the increase in the pro-inflammatory cytokine levels would be lower compared to that in the single high dose exposure group [14
]. To confirm that the animals were in the ET state, we measured the protein levels of inflammatory cytokines after the LPS challenge dose. Although the basal levels in the brain, spleen, and serum of the control groups (SS+S) of F344 and HIV-1Tg rats were similar, the changes in the cytokine and chemokine profiles in response to LPS were different in the HIV-1Tg and F344 rats. We found that, during ET, there was a significantly diminished expression of pro-inflammatory cytokines, such as IL-1β, IFN-γ, and TNF-α, in response to LPS (LL+L) compared to a single exposure of LPS (SS+L).
Of particular interest was the finding that the basal level of IFN-γ in the spleen was lower in the SS+S group of HIV-1Tg rats than of the F344 rats, but was significantly increased in the SS+L and LL+L groups of the HIV-1Tg rats compared to the F344 rats (Table ). A recent study showed that IFN-γ countered ET by facilitating Toll-like receptor (TLR)-induced chromatin remodeling [61
]. ET was prevented in IFN-γ pre-treated primary human monocytes, and production of pro-inflammatory cytokines, such as TNF-α and IL-6, was restored by facilitating TLR-induced chromatin remodeling [61
]. It would be interesting to examine what role IFN-γ plays in the restoration of the production of pro-inflammatory cytokines at the transcriptional level in the HIV-1Tg rat compared to control.
We found that, in both the ET state and after a single exposure to LPS, there was an altered response to LPS in terms of pro-inflammatory cytokine production in the spleen of the HIV-1Tg rats compared to the F344 rats. The expression level of the pro-inflammatory cytokines, IL-1α, IL-1β, and IFN-γ, was 4- to 82-fold greater in the spleen of the HIV-1Tg rats compared to the F344 rats in both the single LPS exposure (SS+L) and ET (LL+L) groups, indicating that the presence of viral proteins may have an effect on innate immune responses. There was also a difference in the expression of cytokine receptors in the brain of the HIV-1Tg rats compared to the F344 rats in response to LPS in both the ET state and after a single exposure to LPS, suggesting that HIV-1 viral proteins may interact with or work through cytokine receptors in the brain. These data warrant further investigation into the neuroimmune effects of HIV-1 viral proteins.
During ET, the production of pro-inflammatory cytokines, such as IL-1β, IL-4, and IL-5, in response to LPS (LL+L) was diminished in contrast to that elicited by a single exposure to the endotoxin (SS+L). This altered response was seen in the spleen and serum in both the HIV-1Tg and F344 ET rats.
The spleen is an immune system organ, and, as such, one would expect that a greater number of cytokine and chemokine genes would be changed in the spleen in response to an immune challenge compared to the brain, and that is what we found in this study. In both the HIV-1Tg and F344 rats, there was a greater response to LPS in the spleen than in the brain.
Chemokines and their receptors have been implicated in the neuropathogenesis of HIV-1 infection [62
]. There were significant differences in chemokine expression in response to LPS in the HIV-1Tg rats compared to the F344 rats. Enhanced levels of Ccl2 in HIV-1 patients have been associated with HIV-1-associated dementia [43
]. Our results indicate that Ccl2 levels were increased to a greater extent (3- to 5-fold) in the brain of the SS+L and LL+L groups of HIV-1Tg rats versus the F344 rats, corroborating the role of upregulation of Ccl2 and its implications on HIV encephalopathy. The levels of the inflammatory CXC chemokines, Cxcl1, Cxcl2, Cxcl10, and Cxcl11, were elevated to a greater extent (0.1- to 40-fold) in the brain of the SS+L and LL+L groups of the F344 rats compared to the HIV-1Tg rats. Ccl2, Ccl7, and Ccl9, levels were increased to a greater extent in the SS+L and LL+L groups of the spleen of the HIV-1Tg rats in comparison to the F344 rats. Cxcl1 and Cxcl2 levels were elevated to a lesser extent in the spleen of the SS+L and LL+L groups of the F344 rats compared to the HIV-1Tg rats.
Chemokines and chemokine receptors define a network throughout the body, playing critical roles in immune and inflammatory responses as well as in many pathological processes, in diseases such as multiple sclerosis, Alzheimer's disease, and HIV/AIDS [63
]. Cxcr4 and Ccr5 are reported to be co-receptors that mediate HIV-1 entry [64
]. In our study, the gene expression of the chemokine receptors, Ccr2, Ccr3, Ccr4, Ccr5, Ccr7, Cxcr3, Ccr10, Ccr3, Cx3cr1, IL-8rβ, and Xcr1, in the spleen of the ET group (LL+L) of F344 rats were down-regulated, whereas those in the HIV-1Tg spleen were not significantly different compared to the control group (SS+S). All these receptors have been shown to function as co-receptors for HIV-1infection in vitro
], which suggests that HIV-1 viral proteins may interact with these chemokine receptors in vivo
. There is also evidence that chemokines and chemokine receptors play an important part in the signaling of neuroprotective effects in the brain [63
In this study, we noted a distinct pattern of cytokine/chemokine expression in the brain, spleen, and serum of the HIV-1Tg and F344 rats in response to LPS, both with and without ET. Identifying these distinct cytokine/chemokine profiles may potentially be useful as indicators of the onset and/or progression of certain disease processes, such as sepsis. Further studies will be done to determine the relationship of viral protein expression to the production of cytokines and chemokines during ET.