LP-BM5 retrovirus infection is an established murine model of HIV infection in humans due to that susceptible strains of mice (such as B6) develop immunodeficiency syndrome. Previously, we have shown that infected B6 mice developed peripheral neuropathy and CD40 knockout (KO) mice displayed less mechanical hypersensitivity (behavioral sign of peripheral neuropathy) post-LP-BM5 infection. The spinal cord viral RNA levels were significantly lower in CD40 KO mice compared to wild type (WT) B6 mice, which is correlated with decreased lumbar spinal cord cytokines, IL-1-beta, IL-6 and IFN-gamma. It is known that LP-BM5 can infect glial cells directly. We hypothesized that glial cells in CD40 KO mice were less susceptible to LP-BM5 infection. To test this hypothesis, primary spinal cord mixed glial cells from both WT and CD40 KO mice were infected with various doses of LP-BM5. Consistent with our in vivo findings, levels of viral RNA are significantly lower in mixed glial cells from CD40 KO mice than that from WT mice. Further, levels of cytokines, IL-1-beta, IL-6, TNF-alpha, IFN-gamma, and IL-12p40 were measured in the supernatants. LP-BM5 only induced minimal increase of these cytokines and there no significant differences between WT and CD40 KO mice were observed. Our data indicate that glial cells lacking CD40 are more resistant to LP-BM5 infection and this resistance does not seem to be due to the decreased production of proinflammatory cytokines by glial cells.
The underlying mechanism of HIV-associated peripheral neuropathy is unclear. C57BL/6 (B6) mice infected with LP-BM5 develop severe immunodeficiency (termed as MAIDS) and peripheral neuropathy. It is known that CD154-CD40 interactions between CD4+ T and B cells are required for the manifestation of MAIDS post-LP-BM5 infection (p.i.) and that HIV encephalitis is associated with increased expression of CD40 on microglia. We aimed to determine whether spinal cord microglial CD40 signaling is involved in LP-BM5-induced peripheral neuropathy. B6-CD40 KO mice were adoptively transferred with either total leukocytes or B cells and examined for mechanical sensitivity, tissue viral loads, and cytokine responses along with the development of MAIDS p.i.. CD40 KO mice transferred either cell population developed MAIDS, the severity of which was correlated with peripheral cytokine responses. CD40 KO mice displayed transient and slightly increased mechanical sensitivity p.i. regardless of cell transfer. In all peripheral tissues examined, increased viral RNA was detected in all infected mice without significant group differences. However, in the lumbar spinal cord, infected wild type B6 mice had the highest viral load and cytokine responses among all groups. Neither type of cell transfer enhanced the LP-BM5-induced increase in viral load and cytokine responses in CD40 KO mice. Together, these data support the involvement of microglial CD40 in LP-BM5-induced peripheral neuropathy.
Intravenous opioid use contributes to 11% of HIV infections in the U.S.A. Morphine suppresses the immune system while exacerbating the progression of HIV. To investigate morphine effects on HIV-associated neurocognitive disorders (HAND), we used the LP-BM5 infection murine AIDS (MAIDS) model. Previously, we found that morphine exposure at 7 wks post-infection (p.i.) enhanced cognitive impairment associated with MAIDS, increased hippocampal viral loads, and decreased CCL5 expression in examined brain regions, without affecting peripheral immunodeficiency. Because many HIV+ patients are exposed to opioids before infection, we examined these parameters after exposing the animals to morphine before and immediately after infection. Male mice were implanted with two placebo or 25mg morphine pellets for 6 days, with infection occurring on post-pellet implantation day 3. At 7 wks p.i., cognitive impairment was assessed by the spontaneous alternation T-maze task. At wk 8, blood, spleen and brain regions (hippocampus, striatum and frontal lobe) were harvested. LP-BM5 induced splenomegaly and higher levels of serum IgG2a and IgM, regardless of morphine treatment. Morphine significantly decreased body weight-normalized spleen weight. Morphine did not alter viral BM5Def RNA in spleen or any brain region examined. Morphine appeared to reduce LP-BM5-induced CCL5 expression in the striatum and frontal lobe. LP-BM5 alone appeared to increase the expression of both IFN-α and IFN-β in the frontal lobe only. Infection or morphine exposure did not affect animals’ performance in the T-maze task. Together with our previous results, these results suggest that timing of morphine exposure can alter the modulation and progression of MAIDS and HAND.
LP-BM5 is a murine retrovirus that induces peripheral neuropathy and AIDS like immune deficiency in B6 mice. Upon infection, primary sensory neurons in the spinal cord express elevated levels of calcitonin gene related protein (CGRP). Previously, we showed that CGRP induced reduction of LP-BM5 viral loads in primary mixed glial cells. The purpose of this current study was to investigate possible CGRP downstream targets that may contribute to its antiretroviral response. We hypothesized that CGRP decreases the viral load in glial cells by modifying the CD40 expression; increasing in CD40 signaling may promote production of cytokines and chemokines with antiretroviral properties. Using CD40 knockout mice we showed that CD40 is required for CGRP mediated antiretroviral response in mixed glia. In microglial cell line, CD40 expression was elevated after treatment with CGRP. Similar effect was seen in microglia from spinal cord derived mixed glial cultures. While LP-BM5 increased microglial CD40 expression gradually over time up to 7 days post-infection in primary mixed glia, CGRP caused an early elevation (24 hr post-infection) of microglial CD40 expression in microglia and stayed relatively steady levels 7 days after infection. CGRP also promoted chemokine CXCL1(KC) production in mixed glia. Our data suggest that activation of CD40 mediated signaling may result in increased production of chemokines that may interfere with retroviral replication. Future studies will be directed to specifically delineate CD40 downstream target involved in antiretroviral response in glial cells.
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