Integrins in muscle disease and repair

Integrin α7β1 plays an important role in maintaining adult skeletal muscle integrity and like dystrophin, provides anchorage and bidirectional signaling as a laminin receptor. The expression of α7β1 integrin was upregulated upon dystrophin deficiency arguing for the molecular compensation and thus considered as potential candidate for treatment of Duchenne Muscular Dystrophy (DMD). The existence of developmentally regulated alternative splice variants makes the α7β1 integrin a complex integrin to study its function in skeletal muscle. In this study we show that increased levels of the adult extracellular variant X2 interfere with muscle integrity, while the presence of embryonic integrin α7 extracellular variant X1 results in normal skeletal muscle architecture. Furthermore, detailed analysis of mdxα7tg mice suggests that overexpression of integrin α7 made no difference on the dystrophic phenotype, in fact mdx α7X2 mice show a more severe phenotype compared to mdx mice. Our study also shed light on the importance of integrin α5 during the development of the skeletal muscle by means of generating conditional knockout (cKO) mice using HSA-Cre and Pax3-Cre promoter systems. Our findings show no obvious difference in the Itga5 cKO when the HSA promoter drives Cre recombinase, however conditional loss under the control of the Pax3 promoter leads perinatal lethality. In addition we investigate the dosage effect of integrin α5 in integrin α7 knockout (KO) mice to understand the cross talk between these two integrins and to correlate with previous data suggesting a gain of function phenotype by that existence of integrin α5 at the myotendinious junction (MTJ) in α7KO muscle (Nawrotzki et al.,2003) From our data we know that gene therapy with integrin α7 is a challenge and is not a suitable alternative to cure dystrophy, at least not in mdx mice, we therefore switch our focus on looking into cell based therapies for DMD by investigating the potential role of perivascular cells (PVCs) using transplantation experiments in mice by artificially inducing muscle damage

The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3 associated phagocytosis

Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP −/- mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality. Abbreviations: CCD: coiled-coil domain; CYBB/NOX2: cytochrome b-245: beta polypeptide; GPT/ALT: glutamic pyruvic transaminase: soluble; LAP: LC3-associated phagocytosis; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NOD: nucleotide-binding oligomerization domain; NADPH: nicotinamide adenine dinucleotide phosphate; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein; SLE: systemic lupus erythematosus; SQSTM1/p62: sequestosome 1; TLR: toll-like receptor; TMEM: transmembrane protein; TRIM: tripartite motif-containing protein; UVRAG: UV radiation resistance associated gene; WD: tryptophan-aspartic acid; WIPI: WD 40 repeat domain: phosphoinositide interacting

ROS-mediated PI3K activation drives mitochondrial transfer from stromal cells to hematopoietic stem cells in response to infection

Hematopoietic stem cells (HSCs) undergo rapid expansion in response to stress stimuli. Here we investigate the bioenergetic processes which facilitate the HSC expansion in response to infection. We find that infection by Gram-negative bacteria drives an increase in mitochondrial mass in mammalian HSCs, which results in a metabolic transition from glycolysis toward oxidative phosphorylation. The initial increase in mitochondrial mass occurs as a result of mitochondrial transfer from the bone marrow stromal cells (BMSCs) to HSCs through a reactive oxygen species (ROS)-dependent mechanism. Mechanistically, ROS-induced oxidative stress regulates the opening of connexin channels in a system mediated by phosphoinositide 3-kinase (PI3K) activation, which allows the mitochondria to transfer from BMSCs into HSCs. Moreover, mitochondria transfer from BMSCs into HSCs, in the response to bacterial infection, occurs before the HSCs activate their own transcriptional program for mitochondrial biogenesis. Our discovery demonstrates that mitochondrial transfer from the bone marrow microenvironment to HSCs is an early physiologic event in the mammalian response to acute bacterial infection and results in bioenergetic changes which underpin emergency granulopoiesis

Foxp3+ follicular regulatory T cells control the germinal center response

Follicular helper (T FH) cells provide crucial signals to germinal center B cells undergoing somatic hypermutation and selection that results in affinity maturation. Tight control of T FH numbers maintains self tolerance. We describe a population of Foxp3 + Blimp-1 + CD4 + T cells constituting 10-25% of the CXCR5 high PD-1 high CD4 + T cells found in the germinal center after immunization with protein antigens. These follicular regulatory T (T FR) cells share phenotypic characteristics with T FH and conventional Foxp3 + regulatory T (T reg) cells yet are distinct from both. Similar to T FH cells, T FR cell development depends on Bcl-6, SLAM-associated protein (SAP), CD28 and B cells; however, T FR cells originate from thymic-derived Foxp3 + precursors, not naive or T FH cells. T FR cells are suppressive in vitro and limit T FH cell and germinal center B cell numbers in vivo. In the absence of T FR cells, an outgrowth of non-antigen-specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, the T FH differentiation pathway is co-opted by T reg cells to control the germinal center response

Foxp3+ follicular regulatory T cells control the germinal center response

<p>Follicular helper (T_FH) cells provide crucial signals to germinal center B cells undergoing somatic hypermutation and selection that results in affinity maturation. Tight control of T_FH numbers maintains self tolerance. We describe a population of Foxp3⁺Blimp-1⁺CD4⁺ T cells constituting 10&#x02013;25% of the CXCR5^highPD-1^highCD4⁺ T cells found in the germinal center after immunization with protein antigens. These follicular regulatory T (T_FR) cells share phenotypic characteristics with T_FH and conventional Foxp3⁺ regulatory T (T_reg) cells yet are distinct from both. Similar to T_FH cells, T_FR cell development depends on Bcl-6, SLAM-associated protein (SAP), CD28 and B cells; however, T_FR cells originate from thymic-derived Foxp3⁺ precursors, not naive or T_FH cells. T_FR cells are suppressive <i>in vitro</i> and limit T_FH cell and germinal center B cell numbers <i>in vivo</i>. In the absence of T_FR cells, an outgrowth of non&#x02013;antigen-specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, the T_FH differentiation pathway is co-opted by T_reg cells to control the germinal center response.</p