Cyclic Adenosine Monophosphate (cAMP)-Dependent Phosphodiesterase Inhibition Promotes Bone Anabolism Through CD8+ T Cell Wnt-10b Production in Mice.

Cyclic adenosine monophosphate (cAMP)-dependent phosphodiesterase (PDE) inhibitors such as pentoxifylline (PTX) suppress cAMP degradation and promote cAMP-dependent signal transduction. PDE inhibitors increase bone formation and bone mass in preclinical models and are used clinically to treat psoriatic arthritis by targeting inflammatory mediators including activated T cells. T cell activation requires two signals: antigen-dependent CD3-activation, which stimulates cAMP production; and CD28 co-stimulation, which downregulates cAMP-signaling, through PDE activation. PDE-inhibitors consequently suppress T cell activation by disrupting CD28 co-stimulation. Interestingly, we have reported that when CD8+ T cells are activated in the absence of CD28 co-stimulation, they secrete Wnt-10b, a bone anabolic Wnt ligand that promotes bone formation. In the present study, we investigated whether the bone anabolic activity of the PDE-inhibitor PTX, has an immunocentric basis, involving Wnt-10b production by CD8+ T cells. When wild-type (WT) mice were administered PTX, biochemical markers of both bone resorption and formation were significantly increased, with net bone gain in the axial skeleton, as quantified by micro-computed tomography (µCT). By contrast, PTX increased only bone resorption in T cell knockout (KO) mice, causing net bone loss. Reconstituting T cell-deficient mice with WT, but not Wnt-10b knockout (KO) CD8+ T cells, rescued bone formation and prevented bone loss. To study the role of cAMP signaling in Wnt-10b expression, reverse-transcription polymerase chain reaction (RT-PCR) and luciferase-reporter assays were performed using primary T cells. PDE inhibitors intensified Wnt-10b promoter activity and messenger RNA (mRNA) accumulation in CD3 and CD28 activated CD8+ T cells. In contrast, inhibiting the cAMP pathway mediators protein kinase A (PKA) and cAMP response element-binding protein (CREB), suppressed Wnt-10b expression by T cells activated in the absence of CD28 co-stimulation. In conclusion, the data demonstrate a key role for Wnt-10b production by CD8+ T cells in the bone anabolic response to PDE-inhibitors and reveal competing T cell-independent pro-resorptive properties of PTX, which dominate under T cell-deficient conditions. Selective targeting of CD8+ T cells by PDE inhibitors may be a beneficial approach for promoting bone regeneration in osteoporotic conditions. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Immune Reconstitution Bone Loss Exacerbates Bone Degeneration Due to Natural Aging in a Mouse Model.

BACKGROUND: Immune reconstitution bone loss (IRBL) is a common side-effect of antiretroviral therapy (ART) in people with human immunodeficiency virus (PWH). Immune reconstitution bone loss acts through CD4+ T-cell/immune reconstitution-induced inflammation and is independent of antiviral regimen. Immune reconstitution bone loss may contribute to the high rate of bone fracture in PWH, a cause of significant morbidity and mortality. Although IRBL is transient, it remains unclear whether bone recovers, or whether it is permanently denuded and further compounds bone loss associated with natural aging. METHODS: We used a validated IRBL mouse model involving T-cell reconstitution of immunocompromised mice. Mice underwent cross-sectional bone phenotyping of femur and/or vertebrae between 6 and 20 months of age by microcomputed tomography (µCT) and quantitative bone histomorphometry. CD4+ T cells were purified at 20 months to quantify osteoclastogenic/inflammatory cytokine expression. RESULTS: Although cortical IRBL in young animals recovered with time, trabecular bone loss was permanent and exacerbated skeletal decline associated with natural aging. At 20 months of age, reconstituted CD4+ T cells express enhanced osteoclastogenic cytokines including RANKL, interleukin (IL)-1ß, IL-17A, and tumor necrosis factor-α, consistent with elevated osteoclast numbers. CONCLUSIONS: Immune reconstitution bone loss in the trabecular compartment is permanent and further exacerbates bone loss due to natural aging. If validated in humans, interventions to limit IRBL may be important to prevent fractures in aging PWH.

Reduced bone formation in males and increased bone resorption in females drive bone loss in hemophilia A mice.

Hemophilia A (HA), a rare X-linked recessive genetic disorder caused by insufficient blood clotting factor VIII, leaves affected individuals susceptible to spontaneous and traumatic hemorrhage. Although males generally exhibit severe symptoms, due to variable X inactivation, females can also be severely impacted. Osteoporosis is a disease of the skeleton predisposing patients to fragility fracture, a cause of significant morbidity and mortality and a common comorbidity in HA. Because the causes of osteoporosis in HA are unclear and in humans confounded by other traditional risk factors for bone loss, in this study, we phenotyped the skeletons of F8 total knockout (F8 TKO) mice, an animal model of severe HA. We found that trabecular bone accretion in the axial and appendicular skeletons of male F8 TKO mice lagged significantly between 2 and 6 months of age, with more modest cortical bone decline. By contrast, in female mice, diminished bone accretion was mostly limited to the cortical compartment. Interestingly, bone loss was associated with a decline in bone formation in male mice but increased bone resorption in female mice, a possible result of sex steroid insufficiency. In conclusion, our studies reveal a sexual dimorphism in the mechanism driving bone loss in male and female F8 TKO mice, preventing attainment of peak bone mass and strength. If validated in humans, therapies aimed at promoting bone formation in males but suppressing bone resorption in females may be indicated to facilitate attainment of peak mass in children with HA to reduce the risk for fracture later in life.

Neutralization of CD40 ligand costimulation promotes bone formation and accretion of vertebral bone mass in mice.

Objective: Immunosuppressive biologics are used in the management of RA and additional immunomodulators are under investigation including modulators of the CD40/CD40 ligand (CD40L) costimulation pathway. Tampering with immune function can have unanticipated skeletal consequences due to disruption of the immuno-skeletal interface, a nexus of shared cells and cytokine effectors serving discrete functions in both immune and skeletal systems. In this study, we examined the effect of MR1, a CD40L neutralizing antibody, on physiological bone remodelling in healthy mice. Methods: Female C57BL6 mice were treated with MR1 and BMD was quantified by dual energy X-ray absorptiometry and indices of trabecular bone structure were quantified by micro-CT. Serum biochemical markers were used to evaluate bone turnover and formation indices by histomorphometry. Results: Unexpectedly, MR1 stimulated significant accretion of BMD and trabecular bone mass in the spine, but not in long bones. Surprisingly, bone accretion was accompanied by a significant increase in bone formation, rather than suppression of bone resorption. Mechanistically, MR1-induced bone accrual was associated with increased Treg development and elevated production of cytotoxic T lymphocyte antigen 4, a costimulation inhibitor that promotes T cell anergy and CD8+ T cell expression of the bone anabolic ligand Wnt-10b. Conclusion: Our studies reveal an unexpected bone anabolic activity of pharmacological CD40L suppression. Therapeutic targeting of the CD40L pathway may indeed have unforeseen consequences for the skeleton, but may also constitute a novel strategy to promote bone formation to ameliorate osteoporotic bone loss and reduce fracture risk in the axial skeleton.

CTLA-4Ig (abatacept) balances bone anabolic effects of T cells and Wnt-10b with antianabolic effects of osteoblastic sclerostin.

Activated lymphocytes promote inflammation and bone destruction in rheumatoid arthritis (RA), making T cells and B cells therapeutic targets. Indeed, pharmacological blockade of CD28 costimulation using CTLA-4Ig (abatacept), approved for amelioration of RA, renders T cells dormant (anergic). CTLA-4Ig also promotes bone accretion in healthy mice; surprisingly, however, this effect is driven exclusively through upregulation of bone formation, rather than anti-inflammatory effects on resorption. In the study presented here, we utilized T cell receptor ß gene and Wnt-10b gene knockout mice to investigate the roles of T cells and Wnt-10b in CTLA-4Ig-induced bone anabolism. Ablation of either T cells or Wnt-10b not only abolished CTLA-4Ig-induced bone anabolism but also, paradoxically, suppressed bone formation leading to bone loss. Stalled bone formation was accompanied by bone marrow stromal cell expression of the Wnt pathway inhibitor sclerostin. Our data suggest that an immunoskeletal pivot may promote or suppress bone formation, depending on the net outcome of CTLA-4Ig action directed independently on T cells and osteoblast-linage cells that counter Wnt-10b-induced bone anabolism, by secretion of sclerostin. While CTLA-4Ig action is tipped in favor of bone formation under physiological conditions, pathological immunodeficiency may lead to suppressed bone formation and skeletal damage.

Homeostatic Expansion of CD4+ T Cells Promotes Cortical and Trabecular Bone Loss, Whereas CD8+ T Cells Induce Trabecular Bone Loss Only.

Background: Bone loss occurs in human immunodeficiency virus (HIV) infection but paradoxically is intensified by HIV-associated antiretroviral therapy (ART), resulting in an increased fracture incidence that is largely independent of ART regimen. Inflammation in the bone microenvironment associated with T-cell repopulation following ART initiation may explain ART-induced bone loss. Indeed, we have reported that reconstitution of CD3+ T cells in immunodeficient mice mimics ART-induced bone loss observed in humans. In this study, we quantified the relative effects of CD4+ and CD8+ T-cell subsets on bone. Methods: T-cell subsets in T-cell receptor ß knockout mice were reconstituted by adoptive transfer with CD4+ or CD8+ T-cells subsets were reconstituted in T-cell receptor ß knockout mice by adoptive transfer, and bone turnover, bone mineral density, and indices of bone structure and turnover were quantified. Results: Repopulating CD4+ but not CD8+ T cells significantly diminished bone mineral density. However, micro-computed tomography revealed robust deterioration of trabecular bone volume by both subsets, while CD4+ T cells additionally induced cortical bone loss. Conclusions: CD4+ T-cell reconstitution, a key function of ART, causes significant cortical and trabecular bone loss. CD8+ T cells may further contribute to trabecular bone loss in some patients with advanced AIDS, in whom CD8+ T cells may also be depleted. Our data suggest that bone densitometry used for assessment of the condition of bone in humans may significantly underestimate trabecular bone damage sustained by ART.

Antiretroviral therapy induces a rapid increase in bone resorption that is positively associated with the magnitude of immune reconstitution in HIV infection.

OBJECTIVE: Antiretroviral therapy (ART) paradoxically intensifies bone loss in the setting of HIV infection. Although the extent of bone loss varies, it occurs with virtually all ART types, suggesting a common pathway that may be aligned with HIV disease reversal. Using an animal model of immunodeficiency we recently demonstrated that immune activation associated with CD4 T-cell reconstitution induces increased production of the osteoclastogenic cytokines RANKL and TNFα by immune cells, driving enhanced bone resorption and loss in bone mineral density. DESIGN: To confirm these findings in humans, we investigated the early kinetics of CD4 T-cell recovery in relation to biomarkers of bone turnover and osteoclastogenic regulators in a prospective 24-week cohort study. METHODS: Clinical data and blood sampling for HIV-RNA PCR, CD4 T-cell counts, bone turnover biomarkers, and osteoclastogenic regulators were obtained from ART-naïve HIV-infected study participants initiating standard doses of lopinavir/ritonavir plus tenofovir disoproxil fumarate/emtricitabine at baseline and at weeks 2, 8, 12, and 24 post ART. RESULTS: C-terminal telopeptide of collagen (CTx) a sensitive biomarker of bone resorption rose by 200% above baseline at week 12, remaining elevated through week 24 (α<0.01), and was associated with significant increases in plasma levels of osteoclastogenic regulators [receptor activator of NF-kB ligand (RANKL), tumor necrosis factor alpha, (TNFα)]. Importantly, the magnitude of CD4 T-cell recovery correlated significantly with CTx (rs = 0.387, α=0.01). CONCLUSION: Our data suggest that ART-induced bone loss occurs early, is aligned with early events of immune reconstitution, and these immune changes provide a unifying mechanism to explain in part the skeletal decline common to all ART.

Role of T-cell reconstitution in HIV-1 antiretroviral therapy-induced bone loss.

HIV infection causes bone loss. We previously reported that immunosuppression-mediated B-cell production of receptor activator of NF-κB ligand (RANKL) coupled with decline in osteoprotegerin correlate with decreased bone mineral density (BMD) in untreated HIV infection. Paradoxically, antiretroviral therapy (ART) worsens bone loss although existing data suggest that such loss is largely independent of specific antiretroviral regimen. This led us to hypothesize that skeletal deterioration following HIV disease reversal with ART may be related to T-cell repopulation and/or immune reconstitution. Here we transplant T cells into immunocompromised mice to mimic ART-induced T-cell expansion. T-cell reconstitution elicits RANKL and TNFα production by B cells and/or T cells, accompanied by enhanced bone resorption and BMD loss. Reconstitution of TNFα- or RANKL-null T-cells and pharmacological TNFα antagonist all protect cortical, but not trabecular bone, revealing complex effects of T-cell reconstitution on bone turnover. These findings suggest T-cell repopulation and/or immune reconstitution as putative mechanisms for bone loss following ART initiation.

Body composition and grip strength are improved in transgenic sickle mice fed a high-protein diet.

Key pathophysiology of sickle cell anaemia includes compensatory erythropoiesis, vascular injury and chronic inflammation, which divert amino acids from tissue deposition for growth/weight gain and muscle formation. We hypothesised that sickle mice maintained on an isoenergetic diet with a high percentage of energy derived from protein (35 %), as opposed to a standard diet with 20 % of energy derived from protein, would improve body composition, bone mass and grip strength. Male Berkeley transgenic sickle mice (S; n 8-12) were fed either 20 % (S20) or 35 % (S35) diets for 3 months. Grip strength (BIOSEB meter) and body composition (dual-energy X-ray absorptiometry scan) were measured. After 3 months, control mice had the highest bone mineral density (BMD) and bone mineral content (BMC) (P < 0·005). S35 mice had the largest increase in grip strength. A two-way ANOVA of change in grip strength (P = 0·043) attributed this difference to genotype (P = 0·025) and a trend in type of diet (P = 0·067). l-Arginine (l-Arg) supplementation of the 20 % diet was explored, as a possible mechanism for improvement obtained with the 35 % diet. Townes transgenic sickle mice (TS; n 6-9) received 0·8, 1·6, 3·2 or 6·4 % l-Arg based on the same protocol and outcome measures used for the S mice. TS mice fed 1·6 % l-Arg for 3 months (TS1.6) had the highest weight gain, BMD, BMC and lean body mass compared with other groups. TS3.2 mice showed significantly more improvement in grip strength than TS0·8 and TS1.6 mice (P < 0·05). In conclusion, the high-protein diet improved body composition and grip strength. Outcomes observed with TS1.6 and TS3.2 mice, respectively, confirm the hypothesis and reveal l-Arg as part of the mechanism.

Bioactive silica nanoparticles reverse age-associated bone loss in mice.

We recently reported that in vitro, engineered 50nm spherical silica nanoparticles promote the differentiation and activity of bone building osteoblasts but suppress bone-resorbing osteoclasts. Furthermore, these nanoparticles promote bone accretion in young mice in vivo. We have now investigated the capacity of these nanoparticles to reverse bone loss in aged mice, a model of human senile osteoporosis. Aged mice received nanoparticles weekly and bone mineral density (BMD), bone structure, and bone turnover were quantified. Our data revealed a significant increase in BMD, bone volume, and biochemical markers of bone formation. Biochemical and histological examinations failed to identify any abnormalities caused by nanoparticle administration. Our studies demonstrate that silica nanoparticles effectively blunt and reverse age-associated bone loss in mice by a mechanism involving promotion of bone formation. The data suggest that osteogenic silica nanoparticles may be a safe and effective therapeutic for counteracting age-associated bone loss. FROM THE CLINICAL EDITOR: Osteoporosis poses a significant problem in the society. Based on their previous in-vitro findings, the authors' group investigated the effects of spherical silica nanoparticles in reversing bone loss in a mouse model of osteoporosis. The results showed that intra-peritoneal injections of silica nanoparticles could increase bone mineral density, with little observed toxic side effects. This novel method may prove important in future therapy for combating osteoporosis.

Acute and chronic B cell depletion disrupts CD4+ and CD8+ T cell homeostasis and expansion during acute viral infection in mice.

B cells provide humoral protection against pathogens and promote cellular immunity through diverse nonclassical effector functions. To assess B cell function in promoting T cell homeostasis, mature B cells were either acutely or chronically depleted in mice using CD20 mAb. Acute B cell depletion in either 2- or 4-mo-old mice significantly reduced spleen and lymph node CD4(+) and CD8(+) T cell numbers, including naive, activated, and Foxp3(+)CD25(+)CD4(+) regulatory T cell subsets. The numbers of IFN-γ- and TNF-α-producing T cells were also significantly reduced. Chronic B cell depletion for 6 mo in aged naive mice resulted in a 40-70% reduction in activated CD4(+) and CD8(+) T cell numbers and 20-50% reductions in IFN-γ-producing T cells. Therefore, B cells were necessary for maintaining naive CD4(+) and CD8(+) T cell homeostasis for subsequent optimal T cell expansion in young and old mice. To determine the significance of this finding, a week of B cell depletion in 4-mo-old mice was followed by acute viral infection with lymphocytic choriomeningitis virus Armstrong. Despite their expansion, activated and cytokine-producing CD4(+) and CD8(+) T cell numbers were still significantly reduced 1 wk later. Moreover, viral peptide-specific CD4(+) and CD8(+) T cell numbers and effector cell development were significantly reduced in mice lacking B cells, whereas lymphocytic choriomeningitis virus titers were dramatically increased. Thus, T cell function is maintained in B cell-depleted mice, but B cells are required for optimal CD4(+) and CD8(+) T cell homeostasis, activation, and effector development in vivo, particularly during responses to acute viral infection.

CTLA-4Ig-induced T cell anergy promotes Wnt-10b production and bone formation in a mouse model.

OBJECTIVE: Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by severe joint erosion and systemic osteoporosis. Chronic T cell activation is a hallmark of RA, and agents that target the CD28 receptor on T cells, which is required for T cell activation, are being increasingly used as therapies for RA and other inflammatory diseases. Lymphocytes play complex roles in the regulation of the skeleton, and although activated T cells and B cells secrete cytokines that promote skeletal decline, under physiologic conditions lymphocytes also have key protective roles in the stabilization of skeletal mass. Consequently, disruption of T cell costimulation may have unforeseen consequences for physiologic bone turnover. This study was undertaken to investigate the impact of pharmacologic CD28 T cell costimulation blockade on physiologic bone turnover and structure. METHODS: C57BL6 mice were treated with CTLA-4Ig, a pharmacologic CD28 antagonist or with irrelevant control antibody (Ig), and serum biochemical markers of bone turnover were quantified by enzyme-linked immunosorbent assay. Bone mineral density and indices of bone structure were further measured by dual x-ray absorptiometry and micro-computed tomography, respectively, and static and dynamic indices of bone formation were quantified using bone histomorphometry. RESULTS: Pharmacologic disruption of CD28 T cell costimulation in mice significantly increased bone mass and enhanced indices of bone structure, a consequence of enhanced bone formation, concurrent with enhanced secretion of the bone anabolic factor Wnt-10b by T cells. CONCLUSION: Inhibition of CD28 costimulation by CTLA-4Ig promotes T cell Wnt-10b production and bone formation and may represent a novel anabolic strategy for increasing bone mass in osteoporotic conditions.

B Cell Production of Both OPG and RANKL is Significantly Increased in Aged Mice.

Aging is a risk factor for osteoclastic bone loss and bone fracture. Receptor activator of NF-κB ligand (RANKL) is the key effector cytokine for osteoclastogenesis and bone resorption, and is moderated by its decoy receptor osteoprotegerin (OPG). The development of an inflammatory environment during aging leads to increased bone resorption and loss of bone mineral density (BMD). Interestingly, animal and clinical studies show that OPG is actually increased in aging but fails to fully compensate for endogenous RANKL. Osteoblast- and B-lineage cells are significant sources of physiological OPG, however osteoblast OPG production declines with age, suggesting that elevated OPG in aging may be a consequence of changes in B cell function. In this study we examined BMD and indices of trabecular bone structure during aging, and B cell production of both RANKL and OPG in young and aged mice. Our data reveal significant loss of BMD and trabecular structure with age commensurate with significantly elevated concentrations of both OPG and RANKL in aged mice, and a decline in B cell populations in aged animals. Taken together our data suggest that B cells may be responsible for the elevated concentrations of OPG during aging and are essential to counteract excessive age-associated bone resorption. Paradoxically, B cells themselves likely contribute RANKL in aging and the loss of B cells with age may further contribute to the imbalance in OPG relative to RANKL that predisposes age-associated bone loss.

Nicotinic acetylcholine receptors are sensors for ethanol in lung fibroblasts.

BACKGROUND: Chronic ethanol (EtOH) abuse in humans is known to independently increase the incidence of and mortality due to acute lung injury in at-risk individuals. However, the mechanisms by which EtOH affects lung cells remain incompletely elucidated. In earlier work, we reported that EtOH increased the expression in lung fibroblasts of fibronectin, a matrix glycoprotein implicated in lung injury and repair. This effect was blocked by α-bungarotoxin, a neurotoxin that binds certain nicotinic acetylcholine receptors (nAChRs) thereby implicating nAChRs in this process. Here, we examine the identity of these receptors. METHODS: Mouse lung fibroblasts were stimulated with EtOH (60 mM) or acetylcholine (100 to 500 µM) and evaluated for the expression of fibronectin and nAChRs. Inhibitors to nAChRs or the antioxidant N-acetyl cysteine (NAC) were used to assess changes in fibronectin expression. Animals exposed to EtOH for up to 6 weeks were used to evaluate the expression of nAChRs in vivo. RESULTS: First, in EtOH-treated fibroblasts, we observed increased expression of α4 and α9 nAChR subunits. Second, we found that acetylcholine, a natural ligand for nAChRs, mimicked the effects of EtOH. Dihydro-ß-erythroidin hydrobromide, a competitive inhibitor of α4 nAChR, blocked the increase in fibronectin expression and cell proliferation. Furthermore, EtOH-induced fibronectin expression was inhibited in cells silenced for α4 nAChR. However, EtOH-treated cells showed increased α-bungarotoxin binding suggesting that α4 nAChR mediates the effects of EtOH via a ligand-independent pathway. Knowing there are several important cysteine residues near the ligand-binding site of α4 nAChRs, we tested the antioxidant NAC and found that it too blocked the induction of fibronectin expression by EtOH. Also, fibroblasts exposed to oxidant stress showed increased fibronectin expression that was blocked with α-bungarotoxin. Finally, we showed increased expression of α4 nAChRs in the lung tissue of mice and rats exposed to EtOH suggesting a role for these receptors in vivo. CONCLUSIONS: Altogether, our observations suggest that α4 nAChRs serve as sensors for EtOH-induced oxidant stress in lung fibroblasts, thereby revealing a new mechanism by which EtOH may affect lung cells and tissue remodeling and pointing to nAChRs as potential targets for intervention.

In utero nicotine exposure promotes M2 activation in neonatal mouse alveolar macrophages.

BACKGROUND: Maternal smoking in utero has been associated with adverse health outcomes including lower respiratory tract infections in infants and children, but the mechanisms underlying these associations continue to be investigated. We hypothesized that nicotine plays a significant role in mediating the effects of maternal tobacco smoke on the function of the neonatal alveolar macrophage (AM), the resident immune cell in the neonatal lung. METHODS: Primary AMs were isolated at postnatal day 7 from a murine model of in utero nicotine exposure. The murine AM cell line MH-S was used for additional in vitro studies. RESULTS: In utero nicotine increased interleukin-13 and transforming growth factor-ß1 (TGFß1) in the neonatal lung. Nicotine-exposed AMs demonstrated increased TGFß1 and increased markers of alternative activation with diminished phagocytic function. However, AMs from mice deficient in the α7 nicotinic acetylcholine receptor (α7 nAChR) had less TGFß1, reduced alternative activation, and improved phagocytic functioning despite similar in utero nicotine exposure. CONCLUSION: In utero nicotine exposure, mediated in part via the α7 nAChR, may increase the risk of lower respiratory tract infections in neonates by changing the resting state of AM toward alternative activation. These findings have important implications for immune responses in the nicotine-exposed neonatal lung.

Predisposition for disrepair in the aged lung.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a devastating progressive lung disease with an average survival of only 3 to 5 years. The mechanisms underlying the initiation and progression of IPF are poorly understood, and treatments available have only modest effect on disease progression. Interestingly, the incidence of IPF is approximately 60 times more common in individuals aged 75 years and older, but the mechanism by which aging promotes fibrosis is unclear. The authors hypothesized that aged lungs have a profibrotic phenotype that render it susceptible to disrepair after injury. METHODS: Young and old mice were treated with bleomycin to examine disrepair in the aged lung. In addition, uninjured young and old mouse lungs were analyzed for transforming growth factor-beta 1 (TGF-ß1) production, extracellular matrix composition and lung fibroblast phenotype. Lung fibroblasts were treated with a DNA methyltransferase inhibitor to examine the potential epigenetic mechanisms involved in age-associated phenotypic alterations. RESULTS: The lungs of old mice showed worse fibrosis after bleomycin-induced injury compared with the lungs from young mice. At baseline, aged lungs expressed a profibrotic phenotype characterized by increased mRNA expression for fibronectin extracellular domain A (Fn-EDA) and the matrix metalloproteinases (MMPs) MMP-2 and MMP-9. Old lungs also expressed higher levels of TGF-ß receptor 1 and TGF-ß1 mRNA, protein and activity as determined by increased Smad3 expression, protein phosphorylation and DNA binding. Lung fibroblasts harvested from aged lungs showed reduced expression of the surface molecule Thy-1, a finding also implicated in lung fibrosis; the latter did not seem related to Thy-1 gene methylation. CONCLUSION: Altogether, aged lungs manifest a profibrotic phenotype characterized by enhanced fibronectin extracellular domain A and MMP expression and increased TGF-ß1 expression and signaling and are populated by Thy-1-negative fibroblasts, all implicated in the pathogenesis of lung fibrosis.

Alterations in the immuno-skeletal interface drive bone destruction in HIV-1 transgenic rats.

Osteoporosis and bone fractures are increasingly recognized complications of HIV-1 infection. Although antiretroviral therapy itself has complex effects on bone turnover, it is now evident that the majority of HIV-infected individuals already exhibit reduced bone mineral density before therapy. The mechanisms responsible are likely multifactorial and have been difficult to delineate in humans. The HIV-1 transgenic rat recapitulates many key features of human AIDS. We now demonstrate that, like their human counterparts, HIV-1 transgenic rats undergo severe osteoclastic bone resorption, a consequence of an imbalance in the ratio of receptor activator of NF-kappaB ligand, the key osteoclastogenic cytokine, to that of its physiological decoy receptor osteoprotegerin. This imbalance stemmed from a switch in production of osteoprotegerin to that of receptor activator of NF-kappaB ligand by B cells, and was further compounded by a significantly elevated number of osteoclast precursors. With the advancing age of individuals living with HIV/AIDS, low bone mineral density associated with HIV infection is likely to collide with the pathophysiology of skeletal aging, leading to increased fracture risk. Understanding the mechanisms driving bone loss in HIV-infected individuals will be critical to developing effective therapeutic strategies.

Polyketal microparticles for therapeutic delivery to the lung.

Inflammation in the setting of interstitial lung disease (ILD) occurs in the distal alveolar spaces of the lung, which presents significant challenges for therapeutic delivery. The development of aerosolizable microparticles from non-immunogenic polymers is needed to enable the clinical translation of numerous experimental therapeutics that require localization to the deep lung and repeated delivery for optimal efficacy. Polyketals (PK), a family of polymers, have several unique properties that make them ideal for lung delivery, specifically their hydrolysis into non-acidic, membrane-permeable compounds and their capacity to form microparticles with the aerodynamic properties needed for aerosolization. In this study, we tested the lung biocompatibility of microparticles created from a polyketal polymer, termed PK3, following intratracheal instillation in comparison to commonly used PLGA microparticles. We furthermore tested the initial efficacy of PK3 microparticles to encapsulate and effectively deliver active superoxide dismutase (SOD), a free radical scavenging enzyme, in a model of lung fibrosis. Our findings indicate that PK3 microparticles display no detectable level of alveolar or airway inflammation, whereas PLGA induced a small inflammatory response. Furthermore, SOD-loaded into PK3 microparticles maintained its activity upon release and, when delivered via PK3 microparticles, inhibited the extent of lung fibrosis.

Extracellular matrix influences alveolar epithelial claudin expression and barrier function.

The lung is dynamically remodeled in response to injury, which alters extracellular matrix composition, and can lead to either healthy or impaired lung regeneration. To determine how changes in extracellular matrix can influence alveolar epithelial barrier function, we examined the expression and function of tight junction proteins by rat alveolar epithelial type II cells cultured on one of three different matrix components: type I collagen or fibronectin, matrix glycoproteins which are highly expressed in injured lungs, or laminin, a basement membrane matrix component. Of note, alveolar epithelial cells cultured for 2 days on fibronectin formed high-resistance barriers and showed continuous claudin-3 and claudin-18 localization to the plasma membrane, as opposed to cells cultured on either type I collagen or laminin, which had low resistance monolayers and had areas of cell-cell contact that were claudin deficient. The barrier formed by cells cultured on fibronectin also had preferential permeability to chloride as compared with sodium. Regardless of the initial matrix composition, alveolar epithelial cells cultured for 5 days formed high-resistance barriers, which correlated with increased claudin-18 localization to the plasma membrane and an increase in zonula occludens-1. Day 5 cells on laminin had significantly higher resistance than cells on either fibronectin or type I collagen. Thus, although alveolar epithelial cells on fibronectin formed rapid barriers, it was at the expense of producing an optimized barrier.

Oxidation of extracellular cysteine/cystine redox state in bleomycin-induced lung fibrosis.

Several lines of evidence indicate that depletion of glutathione (GSH), a critical thiol antioxidant, is associated with the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, GSH synthesis depends on the amino acid cysteine (Cys), and relatively little is known about the regulation of Cys in fibrosis. Cys and its disulfide, cystine (CySS), constitute the most abundant low-molecular weight thiol/disulfide redox couple in the plasma, and the Cys/CySS redox state (E(h) Cys/CySS) is oxidized in association with age and smoking, known risk factors for IPF. Furthermore, oxidized E(h) Cys/CySS in the culture media of lung fibroblasts stimulates proliferation and expression of transitional matrix components. The present study was undertaken to determine whether bleomycin-induced lung fibrosis is associated with a decrease in Cys and/or an oxidation of the Cys/CySS redox state and to determine whether these changes were associated with changes in E(h) GSH/glutathione disulfide (GSSG). We observed distinct effects on plasma GSH and Cys redox systems during the progression of bleomycin-induced lung injury. Plasma E(h) GSH/GSSG was selectively oxidized during the proinflammatory phase, whereas oxidation of E(h) Cys/CySS occurred at the fibrotic phase. In the epithelial lining fluid, oxidation of E(h) Cys/CySS was due to decreased food intake. Thus the data show that decreased precursor availability and enhanced oxidation of Cys each contribute to the oxidation of extracellular Cys/CySS redox state in bleomycin-induced lung fibrosis.