Proinflammatory microenvironments within the intestine regulate the differentiation of tissue-resident CD8⁺ T cells responding to infection.

We report that oral infection with Yersinia pseudotuberculosis results in the development of two distinct populations of pathogen-specific CD8(+) tissue-resident memory T cells (TRM cells) in the lamina propria. CD103(-) T cells did not require transforming growth factor-ß (TGF-ß) signaling but were true resident memory cells. Unlike CD103(+)CD8(+) T cells, which were TGF-ß dependent and were scattered in the tissue, CD103(-)CD8(+) T cells clustered with CD4(+) T cells and CX3CR1(+) macrophages and/or dendritic cells around areas of bacterial infection. CXCR3-dependent recruitment of cells to inflamed areas was critical for development of the CD103(-) population and pathogen clearance. Our studies have identified the 'preferential' development of CD103(-) TRM cells in inflammatory microenvironments within the lamina propria and suggest that this subset has a critical role in controlling infection.

Integrin-α7 signaling regulates connexin 43, M-cadherin, and myoblast fusion.

Regenerative medicine treatments for severe skeletal muscle injuries are limited, resulting in persistent functional deficits. Clinical options include neglecting the wound with the expectation that fibrosis will develop or using an autologous muscle graft with minimal functional improvement. A regenerative matrix can be used, but muscle fiber development on these matrices remains a challenge in vivo. Here, we explored the fundamental mechanisms that mediate cell-substrate signaling and its effect on cell-cell communication during myoblast fusion and tube formation to improve outcomes following implantation of matrices used to stimulate muscle regeneration. We previously reported that integrin-α7 was increased on anisotropic biomaterials, suggesting a role for α7ß1 signaling in myoblast communication via connexin 43 and M-cadherin. Our results demonstrated that α7 silencing blocked expression of myogenic differentiation factor 1 (Myod), myogenin (Myog), myogenic factor 6 (Myf6), myosin heavy chain type 1 (Myh1), and transmembrane protein 8c (Tmem8c), indicating that myoblast fusion was inhibited. Expression of α5 and M-cadherin decreased but ß1 and connexin 43 increased. We examined protein production and observed reduced extracellular-signal regulated kinase 1/2 (ERK) in α7-silenced cells that correlated with upregulation of connexin 43 and M-cadherin, suggesting a compensatory pathway. These results indicate that α7 signaling plays a critical role in ex vivo fusion and implicates a relationship with connexin 43 and M-cadherin.

The Upregulation of Integrin αDß2 (CD11d/CD18) on Inflammatory Macrophages Promotes Macrophage Retention in Vascular Lesions and Development of Atherosclerosis.

Macrophage accumulation is a critical step during development of chronic inflammation, initiating progression of many devastating diseases. Leukocyte-specific integrin αDß2 (CD11d/CD18) is dramatically upregulated on macrophages at inflammatory sites. Previously we found that CD11d overexpression on cell surfaces inhibits in vitro cell migration due to excessive adhesion. In this study, we have investigated how inflammation-mediated CD11d upregulation contributes to macrophage retention at inflammatory sites during atherogenesis. Atherosclerosis was evaluated in CD11d-/-/ApoE-/- mice after 16 wk on a Western diet. CD11d deficiency led to a marked reduction in lipid deposition in aortas and isolated macrophages. Macrophage numbers in aortic sinuses of CD11d-/- mice were reduced without affecting their apoptosis and proliferation. Adoptive transfer of fluorescently labeled wild-type and CD11d-/- monocytes into ApoE-/- mice demonstrated similar recruitment from circulation, but reduced accumulation of CD11d-/- macrophages within the aortas. Furthermore, CD11d expression was significantly upregulated on macrophages in atherosclerotic lesions and M1 macrophages in vitro. Interestingly, expression of the related ligand-sharing integrin CD11b was not altered. This difference defines their distinct roles in the regulation of macrophage migration. CD11d-deficient M1 macrophages demonstrated improved migration in a three-dimensional fibrin matrix and during resolution of peritoneal inflammation, whereas migration of CD11b-/- M1 macrophages was not affected. These results prove the contribution of high densities of CD11d to macrophage arrest during atherogenesis. Because high expression of CD11d was detected in several inflammation-dependent diseases, we suggest that CD11d/CD18 upregulation on proinflammatory macrophages may represent a common mechanism for macrophage retention at inflammatory sites, thereby promoting chronic inflammation and disease development.

Developmental Progression and Interrelationship of Central and Effector Regulatory T Cell Subsets.

Resting central Tregs (cTregs) and activated effector Tregs (eTregs) are required for self-tolerance, but the heterogeneity and relationships within and between phenotypically distinct subsets of cTregs and eTregs are poorly understood. By extensive immune profiling and deep sequencing of TCR-ß V regions, two subsets of cTregs, based on expression of Ly-6C, and three subsets of eTregs, based on distinctive expression of CD62L, CD69, and CD103, were identified. Ly-6C(+) cTregs exhibited lower basal activation, expressed on average lower affinity TCRs, and less efficiently developed into eTregs when compared with Ly-6C(-) cTregs. The dominant TCR Vßs of Ly-6C(+) cTregs were shared by eTregs at a low frequency. A single TCR clonotype was also identified that was largely restricted to Ly-6C(+) cTregs, even under conditions that promoted the development of eTregs. Collectively, these findings indicate that some Ly-6C(+) cTregs may persist as a lymphoid-specific subset, with minimal potential to develop into highly activated eTregs, whereas other cTregs readily develop into eTregs. In contrast, subsets of CD62L(lo) eTregs showed higher clonal expansion and were more highly interrelated than cTreg subsets based on their TCR-ß repertoires, but exhibited varied immune profiles. The CD62L(lo) CD69(-) CD103(-) eTreg subset displayed properties of a transitional intermediate between cTregs and more activated eTreg subsets. Thus, eTreg subsets appear to exhibit substantial flexibility, most likely in response to environmental cues, to adopt defined immune profiles that are expected to optimize suppression of autoreactive T cells.

Osteopontin expression by CD103- dendritic cells drives intestinal inflammation.

Intestinal CD103(-) dendritic cells (DCs) are pathogenic for colitis. Unveiling molecular mechanisms that render these cells proinflammatory is important for the design of specific immunotherapies. In this report, we demonstrated that mesenteric lymph node CD103(-) DCs express, among other proinflammatory cytokines, high levels of osteopontin (Opn) during experimental colitis. Opn expression by CD103(-) DCs was crucial for their immune profile and pathogenicity, including induction of T helper (Th) 1 and Th17 cell responses. Adoptive transfer of Opn-deficient CD103(-) DCs resulted in attenuated colitis in comparison to transfer of WT CD103(-) DCs, whereas transgenic CD103(-) DCs that overexpress Opn were highly pathogenic in vivo. Neutralization of secreted Opn expressed exclusively by CD103(-) DCs restrained disease severity. Also, Opn deficiency resulted in milder disease, whereas systemic neutralization of secreted Opn was therapeutic. We determined a specific domain of the Opn protein responsible for its CD103(-) DC-mediated proinflammatory effect. We demonstrated that disrupting the interaction of this Opn domain with integrin α9, overexpressed on colitic CD103(-) DCs, suppressed the inflammatory potential of these cells in vitro and in vivo. These results add unique insight into the biology of CD103(-) DCs and their function during inflammatory bowel disease.

γδ Intraepithelial Lymphocyte Migration Limits Transepithelial Pathogen Invasion and Systemic Disease in Mice.

BACKGROUND & AIMS: Intraepithelial lymphocytes that express the γδ T-cell receptor (γδ IELs) limit pathogen translocation across the intestinal epithelium by unknown mechanisms. We investigated whether γδ IEL migration and interaction with epithelial cells promote mucosal barrier maintenance during enteric infection. METHODS: Salmonella typhimurium or Toxoplasma gondii were administered to knockout (KO) mice lacking either the T cell receptor δ chain (Tcrd) or CD103, or control TcrdEGFP C57BL/6 reporter mice. Intravital microscopy was used to visualize migration of green fluorescent protein (GFP)-tagged γδ T cells within the small intestinal mucosa of mice infected with DsRed-labeled S typhimurium. Mixed bone marrow chimeras were generated to assess the effects of γδ IEL migration on early pathogen invasion and chronic systemic infection. RESULTS: Morphometric analyses of intravital video microscopy data showed that γδ IELs rapidly localized to and remained near epithelial cells in direct contact with bacteria. Within 1 hour, greater numbers of T gondii or S typhimurium were present within mucosae of mice with migration-defective occludin KO γδ T cells, compared with controls. Pathogen invasion in Tcrd KO mice was quantitatively similar to that in mice with occludin-deficient γδ T cells, whereas invasion in CD103 KO mice, which have increased migration of γδ T cells into the lateral intercellular space, was reduced by 63%. Consistent with a role of γδ T-cell migration in early host defense, systemic salmonellosis developed more rapidly and with greater severity in mice with occludin-deficient γδ IELs, relative to those with wild-type or CD103 KO γδ IELs. CONCLUSIONS: In mice, intraepithelial migration to epithelial cells in contact with pathogens is essential to γδ IEL surveillance and immediate host defense. γδ IEL occludin is required for early surveillance that limits systemic disease.

The α11 integrin mediates fibroblast-extracellular matrix-cardiomyocyte interactions in health and disease.

Excessive cardiac interstitial fibrosis impairs normal cardiac function. We have shown that the α11ß1 (α11) integrin mediates fibrotic responses to glycated collagen in rat myocardium by a pathway involving transforming growth factor-ß. Little is known of the role of the α11 integrin in the developing mammalian heart. Therefore, we examined the impact of deletion of the α11 integrin in wild-type mice and in mice treated with streptozotocin (STZ) to elucidate the role of the α11 integrin in normal cardiac homeostasis and in the pathogenesis of diabetes-related fibrosis. As anticipated, cardiac fibrosis was reduced in α11 integrin knockout mice (α11(-/-); C57BL/6 background) treated with STZ compared with STZ-treated wild-type mice (P < 0.05). Unexpectedly, diastolic function was impaired in both vehicle and STZ-treated α11(-/-) mice, as shown by the decreased minimum rate of pressure change and prolonged time constant of relaxation in association with increased end-diastolic pressure (all P < 0.05 compared with wild-type mice). Accordingly, we examined the phenotype of untreated α11(-/-) mice, which demonstrated a reduced cardiomyocyte cross-sectional cell area and myofibril thickness (all P < 0.05 compared with wild-type mice) and impaired myofibril arrangement. Immunostaining for desmin and connexin 43 showed abnormal intermediate filament organization at intercalated disks and impaired gap-junction development. Overall, deletion of the α11 integrin attenuates cardiac fibrosis in the mammalian mouse heart and reduces ECM formation as a result of diabetes. Furthermore, α11 integrin deletion impairs cardiac function and alters cardiomyocyte morphology. These findings shed further light on the poorly understood interaction between the fibroblast-cardiomyocyte and the ECM.

α1ß1 integrin-mediated adhesion inhibits macrophage exit from a peripheral inflammatory lesion.

Integrins are adhesion molecules critical for the recruitment of leukocytes from blood into peripheral tissues. However, whether integrins are also involved in leukocyte exit from peripheral tissues via afferent lymphatics to the draining lymph node remains poorly understood. In this article, we show that adhesion by the collagen IV-binding integrin α1ß1 unexpectedly inhibited macrophage exit from inflamed skin. We monitored macrophages exiting mouse footpads using a newly developed in situ pulse labeling technique. Blockade of α1ß1 integrin or genetic deletion (Itga1(-/-)) increased macrophage exit efficiency. Chemotaxis assays through collagen IV showed more efficient migration of Itga1(-/-) macrophages relative to wild type. Given that macrophages are key orchestrators of inflammation, α1ß1 integrin adhesion may represent a mechanism for regulating inflammatory responses by controlling macrophage exit or persistence in inflamed tissues.

Functional specialization of islet dendritic cell subsets.

Dendritic cells (DC) play important roles in both tolerance and immunity to ß cells in type 1 diabetes. How and why DC can have diverse and opposing functions in islets remains elusive. To answer these questions, islet DC subsets and their specialized functions were characterized. Under both homeostatic and inflammatory conditions, there were two main tissue-resident DC subsets in islets, defined as CD11b(lo/-)CD103(+)CX3CR1(-) (CD103(+) DC), the majority of which were derived from fms-like tyrosine kinase 3-dependent pre-DC, and CD11b(+)CD103(-)CX3CR1(+) (CD11b(+) DC), the majority of which were derived from monocytes. CD103(+) DC were the major migratory DC and cross-presented islet-derived Ag in the pancreatic draining lymph node, although this DC subset displayed limited phagocytic activity. CD11b(+) DC were numerically the predominant subset (60-80%) but poorly migrated to the draining lymph node. Although CD11b(+) DC had greater phagocytic activity, they poorly presented Ag to T cells. CD11b(+) DC increased in numbers and percentage during T cell-mediated insulitis, suggesting that this subset might be involved in the pathogenesis of diabetes. These data elucidate the phenotype and function of homeostatic and inflammatory islet DC, suggesting differential roles in islet immunity.

Transgenic overexpression of the α7 integrin reduces muscle pathology and improves viability in the dy(W) mouse model of merosin-deficient congenital muscular dystrophy type 1A.

Merosin-deficient congenital muscular dystrophy 1A (MDC1A) is a devastating neuromuscular disease that results in children being confined to a wheelchair, requiring ventilator assistance to breathe and premature death. MDC1A is caused by mutations in the LAMA2 gene, which results in the partial or complete loss of laminin-211 and laminin-221, the major laminin isoforms found in the basal lamina of skeletal muscle. MDC1A patients exhibit reduced α7ß1 integrin; however, it is unclear how the secondary loss of α7ß1 integrin contributes to MDC1A disease progression. To investigate whether restoring α7 integrin expression can alleviate the myopathic phenotype observed in MDC1A, we produced transgenic mice that overexpressed the α7 integrin in the skeletal muscle of the dy(W⁻/⁻) mouse model of MDC1A. Enhanced expression of the α7 integrin restored sarcolemmal localization of the α7ß1 integrin to laminin-α2-deficient myofibers, changed the composition of the muscle extracellular matrix, reduced muscle pathology, maintained muscle strength and function and improved the life expectancy of dy(W⁻/⁻) mice. Taken together, these results indicate that enhanced expression of α7 integrin prevents muscle disease progression through augmentation and/or stabilization of the existing extracellular matrix in laminin-α2-deficient mice, and strategies that increase α7 integrin in muscle might provide an innovative approach for the treatment of MDC1A.

Frontline Science: The expression of integrin αD ß2 (CD11d/CD18) on neutrophils orchestrates the defense mechanism against endotoxemia and sepsis.

Neutrophil-macrophage interplay is a fine-tuning mechanism that regulates the innate immune response during infection and inflammation. Cell surface receptors play an essential role in neutrophil and macrophage functions. The same receptor can provide different outcomes within diverse leukocyte subsets in different inflammatory conditions. Understanding the variety of responses mediated by one receptor is critical for the development of anti-inflammatory treatments. In this study, we evaluated the role of a leukocyte adhesive receptor, integrin αD ß2 , in the development of acute inflammation. αD ß2 is mostly expressed on macrophages and contributes to the development of chronic inflammation. In contrast, we found that αD -knockout dramatically increases mortality in the cecal ligation and puncture sepsis model and LPS-induced endotoxemia. This pathologic outcome of αD -deficient mice is associated with a reduced number of monocyte-derived macrophages and an increased number of neutrophils in their lungs. However, the tracking of adoptively transferred fluorescently labeled wild-type (WT) and αD-/- monocytes in WT mice during endotoxemia demonstrated only a moderate difference between the recruitment of these two subsets. Moreover, the rescue experiment, using i.v. injection of WT monocytes to αD -deficient mice followed by LPS challenge, showed only slightly reduced mortality. Surprisingly, the injection of WT neutrophils to the bloodstream of αD-/- mice markedly increased migration of monocyte-derived macrophage to lungs and dramatically improves survival. αD -deficient neutrophils demonstrate increased necrosis/pyroptosis. αD ß2 -mediated macrophage accumulation in the lungs promotes efferocytosis that reduced mortality. Hence, integrin αD ß2 implements a complex defense mechanism during endotoxemia, which is mediated by macrophages via a neutrophil-dependent pathway.

Lack of α8 integrin leads to morphological changes in renal mesangial cells, but not in vascular smooth muscle cells.

BACKGROUND: Extracellular matrix receptors of the integrin family are known to regulate cell adhesion, shape and functions. The α8 integrin chain is expressed in glomerular mesangial cells and in vascular smooth muscle cells. Mice deficient for α8 integrin have structural alterations in glomeruli but not in renal arteries. For this reason we hypothesized that mesangial cells and vascular smooth muscle cells differ in their respective capacity to compensate for the lack of α8 integrin. RESULTS: Wild type and α8 integrin-deficient mesangial cells varied markedly in cell morphology and expression or localization of cytoskeletal molecules. In α8 integrin-deficient mesangial cells α-smooth muscle actin and CTGF were downregulated. In contrast, there were no comparable differences between α8 integrin-deficient and wild type vascular smooth muscle cells. Expression patterns of integrins were altered in α8 integrin-deficient mesangial cells compared to wild type mesangial cells, displaying a prominent overexpression of α2 and α6 integrins, while expression patterns of the these integrins were not different between wild type and α8 integrin-deficient vascular smooth muscle cells, respectively. Cell proliferation was augmented in α8 integrin-deficient mesangial cells, but not in vascular smooth muscle cells, compared to wild type cells. CONCLUSIONS: Our findings suggest that α8 integrin deficiency has differential effects in mesangial cells and vascular smooth muscle cells. While the phenotype of vascular smooth muscle cells lacking α8 integrin is not altered, mesangial cells lacking α8 integrin differ considerably from wild type mesangial cells which might be a consequence of compensatory changes in the expression patterns of other integrins. This could result in glomerular changes in α8 integrin-deficient mice, while the vasculature is not affected in these mice.

Myotendinous junction defects and reduced force transmission in mice that lack alpha7 integrin and utrophin.

The alpha7beta1 integrin, dystrophin, and utrophin glycoprotein complexes are the major laminin receptors in skeletal muscle. Loss of dystrophin causes Duchenne muscular dystrophy, a lethal muscle wasting disease. Duchenne muscular dystrophy-affected muscle exhibits increased expression of alpha7beta1 integrin and utrophin, which suggests that these laminin binding complexes may act as surrogates in the absence of dystrophin. Indeed, mice that lack dystrophin and alpha7 integrin (mdx/alpha7(-/-)), or dystrophin and utrophin (mdx/utr(-/-)), exhibit severe muscle pathology and die prematurely. To explore the contribution of the alpha7beta1 integrin and utrophin to muscle integrity and function, we generated mice lacking both alpha7 integrin and utrophin. Surprisingly, mice that lack both alpha7 integrin and utrophin (alpha7/utr(-/-)) were viable and fertile. However, these mice had partial embryonic lethality and mild muscle pathology, similar to alpha7 integrin-deficient mice. Dystrophin levels were increased 1.4-fold in alpha7/utr(-/-) skeletal muscle and were enriched at neuromuscular junctions. Ultrastructural analysis revealed abnormal myotendinous junctions, and functional tests showed a ninefold reduction in endurance and 1.6-fold decrease in muscle strength in these mice. The alpha7/utr(-/-) mouse, therefore, demonstrates the critical roles of alpha7 integrin and utrophin in maintaining myotendinous junction structure and enabling force transmission during muscle contraction. Together, these results indicate that the alpha7beta1 integrin, dystrophin, and utrophin complexes act in a concerted manner to maintain the structural and functional integrity of skeletal muscle.

Laminin-111 restores regenerative capacity in a mouse model for alpha7 integrin congenital myopathy.

Mutations in the alpha7 integrin gene cause congenital myopathy characterized by delayed developmental milestones and impaired mobility. Previous studies in dystrophic mice suggest the alpha7beta1 integrin may be critical for muscle repair. To investigate the role that alpha7beta1 integrin plays in muscle regeneration, cardiotoxin was used to induce damage in the tibialis anterior muscle of alpha7 integrin-null mice. Unlike wild-type muscle, which responded rapidly to repair damaged myofibers, alpha7 integrin-deficient muscle exhibited defective regeneration. Analysis of Pax7 and MyoD expression revealed a profound delay in satellite cell activation after cardiotoxin treatment in alpha7 integrin-null animals when compared with wild type. We have recently demonstrated that the muscle of alpha7 integrin-null mice exhibits reduced laminin-alpha2 expression. To test the hypothesis that loss of laminin contributes to the defective muscle regeneration phenotype observed in alpha7 integrin-null mice, mouse laminin-111 (alpha1, beta1, gamma1) protein was injected into the tibialis anterior muscle 3 days before cardiotoxin-induced injury. The injected laminin-111 protein infiltrated the entire muscle and restored myogenic repair and muscle regeneration in alpha7 integrin-null muscle to wild-type levels. Our data demonstrate a critical role for a laminin-rich microenvironment in muscle repair and suggest laminin- 111 protein may serve as an unexpected and novel therapeutic agent for patients with congenital myopathies.

Congenital muscular dystrophies: toward molecular therapeutic interventions.

Congenital muscular dystrophies (CMDs) are a clinically and genetically heterogeneous group of neuromuscular disorders that typically present at birth or in early infancy with hypotonia, weakness, and histologic evidence of a dystrophic myopathy. CMD biochemical types include various abnormalities of alpha-dystroglycan O-mannosyl glycosylation as well as defects in integrin matrix receptors, the extracellular matrix proteins laminin-alpha(2) and collagen VI, nuclear proteins such as lamin A/C, and a protein of the endoplasmic reticulum, selenoprotein N. Current therapies are directed mostly at supportive care; however, recent advances in biotechnology and increased knowledge of the pathophysiology underlying the various CMD types have helped identify potential therapeutic strategies directed at genetic, molecular, and biochemical pathways involved in these disorders. In this article, we review our current understanding of the molecular pathogenesis of several CMD types and how these mechanisms may be therapeutically targeted.

CD103+ cDC1 and endogenous CD8+ T cells are necessary for improved CD40L-overexpressing CAR T cell antitumor function.

While effective in specific settings, adoptive chimeric antigen receptor (CAR) T cell therapy for cancer requires further improvement and optimization. Our previous results show that CD40L-overexpressing CAR T cells mobilize endogenous immune effectors, resulting in improved antitumor immunity. However, the cell populations required for this protective effect remain to be identified. Here we show, by analyzing Batf3-/- mice lacking the CD103+ conventional dendritic cell type 1 (cDC1) subpopulation important for antigen cross-presentation, that CD40L-overexpressing CAR T cells elicit an impaired antitumor response in the absence of cDC1s. We further find that CD40L-overexpressing CAR T cells stimulate tumor-resident CD11b-CD103- double-negative (DN) cDCs to proliferate and differentiate into cDC1s in wild-type mice. Finally, re-challenge experiments show that endogenous CD8+ T cells are required for protective antitumor memory in this setting. Our findings thus demonstrate the stimulatory effect of CD40L-overexpressing CAR T cells on innate and adaptive immune cells, and provide a rationale for using CD40L-overexpressing CAR T cells to improve immunotherapy responses.

Loss of the alpha7 integrin promotes extracellular signal-regulated kinase activation and altered vascular remodeling.

Vascular smooth muscle cell (VSMC) proliferation and migration are underlying factors in the development and progression of cardiovascular disease. Studies have shown that altered expression of vascular integrins and extracellular matrix proteins may contribute to the vascular remodeling observed after arterial injury and during disease. We have recently shown that loss of the alpha7beta1 integrin results in VSMC hyperplasia. To investigate the cellular mechanisms underlying this phenotype, we have examined changes in cell signaling pathways associated with VSMC proliferation. Several studies have demonstrated the mitogen-activated protein kinase signaling pathway is activated in response to vascular injury and disease. In this study, we show that loss of the alpha7 integrin in VSMCs results in activation of the extracellular signal-regulated kinase and translocation of the activated kinase to the nucleus. Forced expression of the alpha7 integrin or use of the mitogen-activated protein kinase kinase 1 inhibitor U0126 in alpha7 integrin-deficient VSMCs suppressed extracellular signal-regulated kinase activation and restored the differentiated phenotype to alpha7 integrin-null cells in a manner dependent on Ras signaling. Alpha7 integrin-null mice displayed profound vascular remodeling in response to injury with pronounced neointimal formation and reduced vascular compliance. These findings demonstrate that the alpha7beta1 integrin negatively regulates extracellular signal-regulated kinase activation and suggests an important role for this integrin as part of a signaling complex regulating VSMC phenotype switching.

Role of integrin alpha8 in murine model of lung fibrosis.

BACKGROUND: Integrin α8 (ITGA8) heterodimerizes with integrin ß1 and is highly expressed in stromal cells of the lung. Platelet-derived growth factor receptor beta (PDGFRß+) cells constitute a major population of contractile myofibroblasts in the lung following bleomycin-induced fibrosis. Integrin α8ß1 is upregulated in fibrotic foci in bleomycin-induced lung injury. However, the functional role of ITGA8 in fibrogenesis has not been characterized. In this study, we examined whether genetic deletion of ITGA8 from PDGFRß+ cells in the lung altered fibrosis. METHODS: Pdgfrb-Cre/+;Itga8flox/- or Pdgfrb-Cre/+;Itga8flox/flox (Cre+) and control mice (Cre-) were used for in vitro and in vivo studies. Primary cultures of PDGFRß+ cells were exposed to TGFß, followed by RNA isolation for qPCR. For in vivo studies, Cre+ and Cre- mice were characterized at baseline and after bleomycin-induced fibrosis. RESULTS: PDGFRß-selected cells from Cre+ animals showed higher levels of Col1a1 expression after treatment with TGFß. However, Cre- and Cre+ animals showed no significant difference in measures of acute lung injury or fibrosis following bleomycin challenge. CONCLUSION: While ITGA8 deletion in lung PDGFRß+ stromal cells showed evidence of greater Col1a1 mRNA expression after TGFß treatment in vitro, no functional difference was detected in vivo.

Cytoplasmic gamma-actin expression in diverse animal models of muscular dystrophy.

We recently showed that cytoplasmic gamma-actin (gamma(cyto)-actin) is dramatically elevated in striated muscle of dystrophin-deficient mdx mice. Here, we demonstrate that gamma(cyto)-actin is markedly increased in golden retriever muscular dystrophy (GRMD), which better recapitulates the dystrophinopathy phenotype in humans. Gamma(cyto)-Actin was also elevated in muscle from alpha-sarcoglycan null mice, but not in several other dystrophic animal models, including mice deficient in beta-sarcoglycan, alpha-dystrobrevin, laminin-2, or alpha7 integrin. Muscle from mice lacking dystrophin and utrophin also expressed elevated gamma(cyto)-actin, which was not restored to normal by transgenic overexpression of alpha7 integrin. However, gamma(cyto)-actin was further elevated in skeletal muscle from GRMD animals treated with the glucocorticoid prednisone at doses shown to improve the dystrophic phenotype and muscle function. These data suggest that elevated gamma(cyto)-actin is part of a compensatory cytoskeletal remodeling program that may partially stabilize dystrophic muscle in some cases where the dystrophin-glycoprotein complex is compromised.

Placental defects in alpha7 integrin null mice.

The alpha7beta1 integrin is a heterodimeric transmembrane receptor that links laminin in the extracellular matrix to the cell cytoskeleton. Loss of the alpha7 integrin chain results in partial embryonic lethality. We have previously shown that alpha7 integrin null embryos exhibit vascular smooth muscle cell defects that result in cerebral vascular hemorrhaging. Since the placenta is highly vascularized, we hypothesized that placental vascular defects in alpha7 integrin null embryos may contribute to the partial embryonic lethality. Placentae from embryonic day (ED) 9.5 and 13.5 alpha7 integrin knockout embryos showed structural defects including infiltration of the spongiotrophoblast layer into the placental labyrinth, a reduction in the placental labyrinth and loss of distinct placental layers. Embryos and placentae that lacked the alpha7 integrin weighed less compared to wild-type controls. Blood vessels within the placental labyrinth of alpha7 integrin null embryos exhibited fewer differentiated vascular smooth muscle cells compared to wild-type. Loss of the alpha7 integrin resulted in altered extracellular matrix deposition and reduced expression of alpha5 integrin. Together our results confirm a role for the alpha7beta1 integrin in placental vascular development and demonstrate for the first time that loss of the alpha7 integrin results in placental defects.