HIV-1-associated neuroinflammation persists even with effective combined anti-retroviral therapy (cART), and is associated with the presence of activated monocytes/macrophages within the CNS. In order to infiltrate the CNS, monocytes transmigrate across the selectively permeable blood brain barrier (BBB), which is compromised during HIV infection. Interestingly, platelet-derived excess soluble CD40L (sCD40L) found in the plasma and cerebrospinal fluid (CSF) of HIV-1 infected individuals with cognitive impairment has previously been implicated in increased BBB permeability. Here we show that sCD40L also promotes the formation of complexes between inflammatory monocytes and activated platelets (PMCs), which are detected by flow cytometry as monocytes that express excess of CD61, a platelet marker and that these complexes are increased in individuals with HIV infection. PMCs exhibit an enhanced ability to adhere to human brain microvascular endothelial cells as compared to monocytes alone and migrate across transendothelial barrier. These complexes can be found marginalized in the lumen of post-capillary venules in post-mortem brain tissue derived from cases of HIV-1-associated encephalitis (HIV-E). The extravasation of monocytes across the brain endothelium may exacerbate neuroinflammation, indicating that enhancing this event via platelet interaction may be a contributing factor in the development of cognitive impairment. Thus, dampening platelet activation, and in turn PMC formation, with anti-platelet agents may prove beneficial in developing adjunctive therapies for use in combination with cART in an effort to reduce HIV-1-associated neurological deficit

Despite the use of anti-retroviral therapies, a majority of HIV-infected individuals still develop HIV-Associated Neurocognitive Disorders (HAND), indicating that host inflammatory mediators, in addition to viral proteins, may be contributing to these disorders. Consistently, we have previously shown that levels of the inflammatory mediator soluble CD40L (sCD40L) are elevated in the circulation of HIV-infected, cognitively impaired individuals as compared to their infected, non-impaired counterparts. Recent studies from our group suggest a role for the CD40/CD40L dyad in blood brain barrier (BBB) permeability and interestingly, sCD40L is thought to regulate BBB permeability in other inflammatory disorders of the CNS. Using complementary multiphoton microscopy and quantitative analyses in wild-type and CD40L deficient mice, we now reveal that the HIV transactivator of transcription (Tat) can induce BBB permeability in a CD40L-dependent manner. This permeability of the BBB was found to be the result of aberrant platelet activation induced by Tat, since depletion of platelets prior to treatment reversed Tat-induced BBB permeability. Furthermore, Tat treatment led to an increase in granulocyte antigen 1 (Gr1) positive monocytes, indicating an expansion of the inflammatory subset of cells in these mice, which were found to adhere more readily to the brain microvasculature in Tat treated animals. Exploring the mechanisms by which the BBB becomes compromised during HIV infection has the potential to reveal novel therapeutic targets, thereby aiding in the development of adjunct therapies for the management of HAND, which are currently lacking.

Activated platelets form transient aggregates with monocytes in circulation and have a half-life of approximately 30–60 minutes. These complexes are increased in various inflammatory conditions and are an early marker of myocardial infarction. HIV-1 infection is associated with chronic inflammation, and increased CD16+ inflammatory monocytes have been observed in these individuals, probably as a result of increased interaction with platelets. However, narrow detection period and platelet activation during sample processing pose significant problems in detecting platelet-monocyte complexes (PMCs). A method was standardized addressing these difficulties, to enumerate PMCs involving CD16+ or CD16− monocytes in whole blood using flow cytometry. Blood collected from healthy individuals was treated with either collagen (for platelet activation) or LPS (for monocyte activation) and subsequently used to study effect of these treatments on PMC formation. This method was also validated for the ex vivo quantitation of PMCs in blood obtained from persons infected with HIV. The in vitro results demonstrated that platelet activation, but not monocyte activation, resulted in significant increase in PMC formation. There was a significant increase in CD16+ PMCs and platelet activation, in samples obtained from persons infected with HIV as compared to those without HIV infection. Furthermore, PMC percentages correlated positively with platelet activation. These findings improve the ability to detect PMCs and shed light on HIV pathogenesis.

Organomodified nanoclays (ONCs) are increasingly used as filler materials to improve nanocomposite strength, wettability, flammability, and durability. However, pulmonary risks associated with exposure along their chemical lifecycle are unknown. This study's objective was to compare pre- and post-incinerated forms of uncoated and organomodified nanoclays for potential pulmonary inflammation, toxicity, and systemic blood response. Mice were exposed via aspiration to low (30 μg) and high (300 μg) doses of preincinerated uncoated montmorillonite nanoclay (CloisNa), ONC (Clois30B), their respective incinerated forms (I-CloisNa and I-Clois30B), and crystalline silica (CS). Lung and blood tissues were collected at days 1, 7, and 28 to compare toxicity and inflammation indices. Well-dispersed CloisNa caused a robust inflammatory response characterized by neutrophils, macrophages, and particle-laden granulomas. Alternatively, Clois30B, I-Clois30B, and CS high-dose exposures elicited a low grade, persistent inflammatory response. High-dose Clois30B exposure exhibited moderate increases in lung damage markers and a delayed macrophage recruitment cytokine signature peaking at day 7 followed by a fibrotic tissue signature at day 28, similar to CloisNa. I-CloisNa exhibited acute, transient inflammation with quick recovery. Conversely, high-dose I-Clois30B caused a weak initial inflammatory signal but showed comparable pro-inflammatory signaling to CS at day 28. The data demonstrate that ONC pulmonary toxicity and inflammatory potential relies on coating presence and incineration status in that coated and incinerated nanoclay exhibited less inflammation and granuloma formation than pristine montmorillonite. High doses of both pre- and post-incinerated ONC, with different surface morphologies, may harbor potential pulmonary health hazards over long-term occupational exposures.

CD40 ligand (CD40L) is a type II membrane glycoprotein of the tumor necrosis factor (TNF) family that is found on activated T cells, B cells, and platelets. We previously reported that the soluble form of this inflammatory mediator (sCD40L) is elevated in the plasma and cerebrospinal fluid of HIV-1 infected, cognitively impaired individuals. Here we demonstrate that the mood-stabilizing drug valproic acid (VPA) reduces sCD40L levels in plasma samples of HIV-1 infected patients (n=23) and in washed human platelets, which are the main source of circulating sCD40L. VPA also inhibited HIV-1 transactivator of transcription (Tat)-induced release of sCD40L and platelet factor 4 in C57BL/6 mice. The mechanism by which VPA was able to do so was investigated and herein we demonstrate that VPA, a known glycogen synthase kinase 3 beta (GSK3β) inhibitor, blocks platelet activating factor (PAF)-induced activation of GSK3β in platelets in a manner that alters sCD40L release from platelets. These data reveal that VPA has anti-platelet activity, and convey important implications for the potential of VPA as an adjunct therapy not only for cognitively impaired patients with HIV-1 infection, but also numerous inflammatory diseases for which such anti-platelet therapies are currently lacking.