Sex-Based Disparities in Leukocyte Migration and Activation in Response to Inhalation Lung Injury: Role of SDF-1/CXCR4 Signaling.

The aim of the study was to determine whether sex-related differences exist in immune response to inhalation lung injury. C57BL/6 mice were exposed to Cl2 gas (500 ppm for 15, 20, or 30 min). Results showed that male mice have higher rates of mortality and lung injury than females. The binding of the chemokine ligand C-X-C motif chemokine 12 (CXCL12), also called stromal-derived-factor-1 (SDF-1), to the C-X-C chemokine receptor type 4 (CXCR4) on lung cells promotes the migration of leukocytes from circulation to lungs. Therefore, the hypothesis was that elevated SDF-1/CXCR4 signaling mediates exaggerated immune response in males. Plasma, blood leukocytes, and lung cells were collected from mice post-Cl2 exposure. Plasma levels of SDF-1 and peripheral levels of CXCR4 in lung cells were higher in male vs. female mice post-Cl2 exposure. Myeloperoxidase (MPO) and elastase activity was significantly increased in leukocytes of male mice exposed to Cl2. Lung cells were then ex vivo treated with SDF-1 (100 ng/mL) in the presence or absence of the CXCR4 inhibitor, AMD3100 (100 nM). SDF-1 significantly increased migration, MPO, and elastase activity in cells obtained from male vs. female mice post-Cl2 exposure. AMD3100 attenuated these effects, suggesting that differential SDF-1/CXCR4 signaling may be responsible for sex-based disparities in the immune response to inhalation lung injury.

High Heme and Low Heme Oxygenase-1 Are Associated with Mast Cell Activation/Degranulation in HIV-Induced Chronic Widespread Pain.

An overwhelming number of people with HIV (PWH) experience chronic widespread pain (CWP) throughout their lifetimes. Previously, we demonstrated that PWH with CWP have increased hemolysis and attenuated heme oxygenase 1 (HO-1) levels. HO-1 degrades reactive, cell-free heme into antioxidants like biliverdin and carbon monoxide (CO). We found that high heme or low HO-1 caused hyperalgesia in animals, likely through multiple mechanisms. In this study, we hypothesized that high heme or low HO-1 caused mast cell activation/degranulation, resulting in the release of pain mediators like histamine and bradykinin. PWH who self-report CWP were recruited from the University of Alabama at Birmingham HIV clinic. Animal models included HO-1-/- mice and hemolytic mice, where C57BL/6 mice were injected intraperitoneally with phenylhydrazine hydrochloride (PHZ). Results demonstrated that plasma histamine and bradykinin were elevated in PWH with CWP. These pain mediators were also high in HO-1-/- mice and in hemolytic mice. Both in vivo and in vitro (RBL-2H3 mast cells), heme-induced mast cell degranulation was inhibited by treatment with CORM-A1, a CO donor. CORM-A1 also attenuated mechanical and thermal (cold) allodynia in hemolytic mice. Together, the data suggest that mast cell activation secondary to high heme or low HO-1 seen in cells and animals correlates with elevated plasma levels of heme, histamine, and bradykinin in PWH with CWP.

Heme-Induced Macrophage Phenotype Switching and Impaired Endogenous Opioid Homeostasis Correlate with Chronic Widespread Pain in HIV.

Chronic widespread pain (CWP) is associated with a high rate of disability and decreased quality of life in people with HIV-1 (PWH). We previously showed that PWH with CWP have increased hemolysis and elevated plasma levels of cell-free heme, which correlate with low endogenous opioid levels in leukocytes. Further, we demonstrated that cell-free heme impairs ß-endorphin synthesis/release from leukocytes. However, the cellular mechanisms by which heme dampens ß-endorphin production are inconclusive. The current hypothesis is that heme-dependent TLR4 activation and macrophage polarization to the M1 phenotype mediate this phenomenon. Our novel findings showed that PWH with CWP have elevated M1-specific macrophage chemokines (ENA-78, GRO-α, and IP-10) in plasma. In vitro, hemin-induced polarization of M0 and M2 macrophages to the M1 phenotype with low ß-endorphins was mitigated by treating cells with the TLR4 inhibitor, TAK-242. Similarly, in vivo phenylhydrazine hydrochloride (PHZ), an inducer of hemolysis, injected into C57Bl/6 mice increased the M1/M2 cell ratio and reduced ß-endorphin levels. However, treating these animals with the heme-scavenging protein hemopexin (Hx) or TAK-242 reduced the M1/M2 ratio and increased ß-endorphins. Furthermore, Hx attenuated heme-induced mechanical, heat, and cold hypersensitivity, while TAK-242 abrogated hypersensitivity to mechanical and heat stimuli. Overall, these results suggest that heme-mediated TLR4 activation and M1 polarization of macrophages correlate with impaired endogenous opioid homeostasis and hypersensitivity in people with HIV.

Overexpression of human apolipoprotein A-I preserves cognitive function and attenuates neuroinflammation and cerebral amyloid angiopathy in a mouse model of Alzheimer disease.

To date there is no effective therapy for Alzheimer disease (AD). High levels of circulating high density lipoprotein (HDL) and its main protein, apolipoprotein A-I (apoA-I), reduce the risk of cardiovascular disease. Clinical studies show that plasma HDL cholesterol and apoA-I levels are low in patients with AD. To investigate if increasing plasma apoA-I/HDL levels ameliorates AD-like memory deficits and amyloid-ß (Aß) deposition, we generated a line of triple transgenic (Tg) mice overexpressing mutant forms of amyloid-ß precursor protein (APP) and presenilin 1 (PS1) as well as human apoA-I (AI). Here we show that APP/PS1/AI triple Tg mice have a 2-fold increase of plasma HDL cholesterol levels. When tested in the Morris water maze for spatial orientation abilities, whereas APP/PS1 mice develop age-related learning and memory deficits, APP/PS1/AI mice continue to perform normally during aging. Interestingly, no significant differences were found in the total level and deposition of Aß in the brains of APP/PS1 and APP/PS1/AI mice, but cerebral amyloid angiopathy was reduced in APP/PS1/AI mice. Also, consistent with the anti-inflammatory properties of apoA-I/HDL, glial activation was reduced in the brain of APP/PS1/AI mice. In addition, Aß-induced production of proinflammatory chemokines/cytokines was decreased in mouse organotypic hippocampal slice cultures expressing human apoA-I. Therefore, we conclude that overexpression of human apoA-I in the circulation prevents learning and memory deficits in APP/PS1 mice, partly by attenuating neuroinflammation and cerebral amyloid angiopathy. These findings suggest that elevating plasma apoA-I/HDL levels may be an effective approach to preserve cognitive function in patients with AD.

Intake of sucrose-sweetened water induces insulin resistance and exacerbates memory deficits and amyloidosis in a transgenic mouse model of Alzheimer disease.

Compelling evidence indicates that excess consumption of sugar-sweetened beverages plays an important role in the epidemic of obesity, a major risk factor for type 2 diabetes mellitus. Type 2 diabetes mellitus has been associated with a higher incidence of Alzheimer disease (AD). High fat diets promote AD-like pathology in mice. It is not known whether consumption of excess sugar as in calorically sweetened beverages with an otherwise normal diet affects the development of AD. In the present study, we provided 10% sucrose-sweetened water to a transgenic mouse model of AD with a normal rodent diet. Compared with the control mice with no sucrose added in the water, the sucrose group gained more body weight and developed glucose intolerance, hyperinsulinemia, and hypercholesterolemia. These metabolic changes were associated with the exacerbation of memory impairment and a 2-3-fold increase in insoluble amyloid-beta protein levels and deposition in the brain. We further showed that the levels of expression and secretase-cleaved products of amyloid-beta precursor protein were not affected by sucrose intake. The steady-state levels of insulin-degrading enzyme did not change significantly, whereas there was a 2.5-fold increase in brain apoE levels. Therefore, we concluded that the up-regulation of apoE accelerated the aggregation of Abeta, resulting in the exacerbation of cerebral amyloidosis in sucrose-treated mice. These data underscore the potential role of dietary sugar in the pathogenesis of AD and suggest that controlling the consumption of sugar-sweetened beverages may be an effective way to curtail the risk of developing AD.

Anti-Abeta single-chain antibody delivery via adeno-associated virus for treatment of Alzheimer's disease.

Immunization of mouse models of Alzheimer disease (AD) with amyloid-peptide (Abeta) reduces Abeta deposits and attenuates their memory and learning deficits. Recent clinical trials were halted due to meningoencephalitis, presumably induced by T cell mediated and/or Fc-mediated immune responses. Because injection of anti-Abeta F(ab')(2) antibodies also induces clearance of amyloid plaques in AD mouse models, we have tested a novel gene therapy modality where an adeno-associated virus (AAV) encoding anti-Abeta single-chain antibody (scFv) is injected into the corticohippocampal regions of AD mouse models. One year after injection, expression of scFv was readily detectable in the neurons of the hippocampus without discernible neurotoxicity. AD mouse models subjected to AAV injection had much less amyloid deposits at the injection sites than the mouse models subjected to PBS injection. Because the scFv lacks the Fc portion of the immunoglobulin molecule, this modality may be a feasible solution for AD without eliciting inflammation.

Amelioration of amyloid load by anti-Abeta single-chain antibody in Alzheimer mouse model.

Parenteral immunization of transgenic mouse models of Alzheimer disease (AD) with synthetic amyloid beta-peptide (Abeta) prevented or reduced Abeta deposits and attenuated their memory and learning deficits. A clinical trial of immunization with synthetic Abeta, however, was halted due to brain inflammation, presumably induced by a toxic Abeta, T-cell- and/or Fc-mediated immune response. Another issue relating to such immunizations is that some AD patients may not be able to raise an adequate immune response to Abeta vaccination due to immunological tolerance or age-associated decline. Because peripheral administration of antibodies against Abeta also induced clearance of amyloid plaques in the model mice, injection of humanized Abeta antibodies has been proposed as a possible therapy for AD. By screening a human single-chain antibody (scFv) library for Abeta immunoreactivity, we have isolated a scFv that specifically reacts with oligomeric Abeta as well as amyloid plaques in the brain. The scFv inhibited Abeta amyloid fibril formation and Abeta-mediated cytotoxicity in vitro. We have tested the efficacy of the human scFv in a mouse model of AD (Tg2576 mice). Relative to control mice, injections of the scFv into the brain of Tg2576 mice reduced Abeta deposits. Because scFvs lack the Fc portion of the immunoglobulin molecule, human scFvs against Abeta may be useful to treat AD patients without eliciting brain inflammation.

Induction of a Th2 immune response by co-administration of recombinant adenovirus vectors encoding amyloid beta-protein and GM-CSF.

Lines of experimental evidence indicate that induction of humoral immune responses in transgenic mouse models of Alzheimer disease (AD) by repeated injection of synthetic amyloid beta-protein (Abeta) is effective in prevention and clearance of deposits of Abeta aggregates in the brain of the mice. We have tested a non-injection modality whereby replication-defective adenovirus vectors encoding Abeta or the 99-amino acid carboxyl terminal fragment of Abeta precursor were intranasally administered to mice to elicit immune responses against Abeta. When mice were immunized only with the adenovirus vectors, immune responses against Abeta were negligible. By co-immunization with an adenovirus vector encoding granulocyte-macrophage colony stimulating factor (GM-CSF), the adenovirus vector encoding Abeta effectively elicited an immune response against Abeta. Immunoglobulin isotyping demonstrated a predominant IgG1 and IgG2b response, suggesting a Th2 anti-inflammatory type. Thus, adjuvantation is essential for induction of an immune response against Abeta by adenovirus-mediated nasal vaccination.

Immunization of Alzheimer model mice with adenovirus vectors encoding amyloid beta-protein and GM-CSF reduces amyloid load in the brain.

Induction of anti-amyloid beta-protein (Abeta) antibodies in transgenic mouse models of Alzheimer disease (AD) by repeated injection of synthetic Abeta was shown to be effective in preventing and removing deposition of Abeta aggregates in the brain. Here, we have tested a non-invasive modality whereby a replication-defective adenovirus vector encoding Abeta was intranasally administered to mice to elicit immune responses against Abeta. Intranasal immunization only with the adenovirus vector failed to induce significant immune responses. When an adenovirus vector encoding granulocyte/macrophage-colony stimulating factor (GM-CSF) was used as an adjuvant in conjunction with the adenovirus encoding Abeta, a marked immune response was elicited against Abeta. Immunoglobulin isotyping revealed that the induced anti-Abeta antibodies are predominantly of the IgG2b and IgG1 isotypes, suggesting a Th-2 anti-inflammatory type. Furthermore, amyloid load in the brain of AD model mice (Tg2576) vaccinated with adenovirus vectors encoding Abeta and GM-CSF was much smaller than that in control Tg2576 mice. Thus, intranasal administration of adenovirus vectors encoding Abeta and GM-CSF may be effective in prevention and treatment of AD.