Differential adapter recruitment by TLR2 co-receptors.

TLR2 heterodimers with TLR1 or TLR6 recognize distinct pathogen-associated molecules such as tri- and di-acylated lipopeptides. The activated TLR2 heterodimers recruit Toll-IL-1R domain- (TIR-) containing adapter proteins, TIRAP and MyD88, through the receptor TIR domains. Molecular recognition mechanisms responsible for agonist-driven, TIR domain-mediated receptor-adapter interactions as well as the structure of resultant signaling complexes remain unknown. We previously reported that the cell-permeable peptide derived from helix D of TLR2 TIR (2R9) specifically binds TIRAP in vitro and in cells and thereby inhibits TIRAP-dependent TLR signaling. This study demonstrates that cell-permeable peptides from D helix of TLR1 or TLR6, peptides 1R9 and 6R9 respectively, inhibit signaling mediated by cognate TLR2 co-receptors. Interestingly, 1R9 and 6R9 bind different TLR2 adapters, as they selectively bind MyD88 and TIRAP TIR, respectively. Both peptides block the agonist-induced co-immunoprecipitation (co-IP) of TLR2 with TIRAP or MyD88, but not TLR2 co-IP with co-receptors. Our data suggest that D helices of TLR1 and TLR6 TIR domains are adapter recruitment sites in both co-receptors; yet the sites recruit different adapters. The D helix in TLR1 is the MyD88 docking site, whereas in TLR6 this site recruits TIRAP.

A Decoy Peptide that Disrupts TIRAP Recruitment to TLRs Is Protective in a Murine Model of Influenza.

Toll-like receptors (TLRs) activate distinct, yet overlapping sets of signaling molecules, leading to inflammatory responses to pathogens. Toll/interleukin-1 receptor (TIR) domains, present in all TLRs and TLR adapters, mediate protein interactions downstream of activated TLRs. A peptide library derived from TLR2 TIR was screened for inhibition of TLR2 signaling. Cell-permeable peptides derived from the D helix and the segment immediately N-terminal to the TLR2 TIR domain potently inhibited TLR2-mediated cytokine production. The D-helix peptide, 2R9, also potently inhibited TLR4, TLR7, and TLR9, but not TLR3 or TNF-α signaling. Cell imaging, co-immunoprecipitation, and in vitro studies demonstrated that 2R9 preferentially targets TIRAP. 2R9 diminished systemic cytokine responses elicited in vivo by synthetic TLR2 and TLR7 agonists; it inhibited the activation of macrophages infected with influenza strain A/PR/8/34 (PR8) and significantly improved the survival of PR8-infected mice. Thus, 2R9 represents a TLR-targeting agent that blocks protein interactions downstream of activated TLRs.

Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain.

Toll/IL-1R resistance (TIR) domain-containing adapter-inducing IFN-ß (TRIF) is a Toll-like receptor (TLR) adapter that mediates MyD88-independent induction of type I interferons through activation of IFN regulatory factor 3 and NFκB. We have examined peptides derived from the TRIF TIR domain for ability to inhibit TLR4. In addition to a previously identified BB loop peptide (TF4), a peptide derived from putative helix B of TRIF TIR (TF5) strongly inhibits LPS-induced cytokine and MAPK activation in wild-type cells. TF5 failed to inhibit LPS-induced cytokine and kinase activation in TRIF-deficient immortalized bone-marrow-derived macrophage, but was fully inhibitory in MyD88 knockout cells. TF5 does not block macrophage activation induced by TLR2, TLR3, TLR9, or retinoic acid-inducible gene 1/melanoma differentiation-associated protein 5 agonists. Immunoprecipitation assays demonstrated that TF4 binds to TLR4 but not TRIF-related adaptor molecule (TRAM), whereas TF5 binds to TRAM strongly and TLR4 to a lesser extent. Although TF5 prevented coimmunoprecipitation of TRIF with both TRAM and TLR4, site-directed mutagenesis of the TRIF B helix residues affected TRIF-TRAM coimmunoprecipitation selectively, as these mutations did not block TRIF-TLR4 association. These results suggest that the folded TRIF TIR domain associates with TRAM through the TRIF B helix region, but uses a different region for TRIF-TLR4 association. The B helix peptide TF5, however, can associate with either TRAM or TLR4. In a mouse model of TLR4-driven inflammation, TF5 decreased plasma cytokine levels and protected mice from a lethal LPS challenge. Our data identify TRIF sites that are important for interaction with TLR4 and TRAM, and demonstrate that TF5 is a potent TLR4 inhibitor with significant potential as a candidate therapeutic for human sepsis.

Targeting Toll-like receptor (TLR) signaling by Toll/interleukin-1 receptor (TIR) domain-containing adapter protein/MyD88 adapter-like (TIRAP/Mal)-derived decoy peptides.

Toll/interleukin-1 receptor (TIR) domain-containing adapter protein/MyD88 adapter-like (TIRAP/Mal) is an adapter protein that facilitates recruitment of MyD88 to TLR4 and TLR2 signaling complexes. We previously generated a library of cell-permeating TLR4 TIR-derived decoy peptides fused to the translocating segment of the Drosophila Antennapedia homeodomain and examined each peptide for the ability to inhibit TLR4 signaling (Toshchakov, V. Y., Szmacinski, H., Couture, L. A., Lakowicz, J. R., and Vogel, S. N. (2011) J. Immunol. 186, 4819-4827). We have now expanded this study to test TIRAP decoy peptides. Five TIRAP peptides, TR3 (for TIRAP region 3), TR5, TR6, TR9, and TR11, inhibited LPS-induced cytokine mRNA expression and MAPK activation. Inhibition was confirmed at the protein level; select peptides abolished the LPS-induced cytokine production measured in cell culture 24 h after a single treatment. Two of the TLR4 inhibitory peptides, TR3 and TR6, also inhibited cytokine production induced by a TLR2/TLR1 agonist, S-(2,3-bis(palmitoyloxy)-(2R,2S)-propyl)-N-palmitoyl-(R)-Cys-Ser-Lys(4)-OH; however, a higher peptide concentration was required to achieve comparable inhibition of TLR2 versus TLR4 signaling. Two TLR4 inhibitory peptides, TR5 and TR6, were examined for the ability to inhibit TLR4-driven cytokine induction in mice. Pretreatment with either peptide significantly reduced circulating TNF-α and IL-6 in mice following LPS injection. This study has identified novel TLR inhibitory peptides that block cellular signaling at low micromolar concentrations in vitro and in vivo. Comparison of TLR4 inhibition by TLR4 and TIRAP TIR-derived peptides supports the view that structurally diverse regions mediate functional interactions of TIR domains.

Distinct effects of contraction-induced injury in vivo on four different murine models of dysferlinopathy.

Mutations in the DYSF gene, encoding dysferlin, cause muscular dystrophies in man. We compared 4 dysferlinopathic mouse strains: SJL/J and B10.SJL-Dysf(im)/AwaJ (B10.SJL), and A/J and B6.A-Dysf(prmd)/GeneJ (B6.A/J). The former but not the latter two are overtly myopathic and weaker at 3 months of age. Following repetitive large-strain injury (LSI) caused by lengthening contractions, all except B6.A/J showed ~40% loss in contractile torque. Three days later, torque in SJL/J, B10.SJL and controls, but not A/J, recovered nearly completely. B6.A/J showed ~30% torque loss post-LSI and more variable recovery. Pre-injury, all dysferlinopathic strains had more centrally nucleated fibers (CNFs) and all but A/J showed more inflammation than controls. At D3, all dysferlinopathic strains showed increased necrosis and inflammation, but not more CNFs; controls were unchanged. Dystrophin-null DMD(mdx) mice showed more necrosis and inflammation than all dysferlin-nulls. Torque loss and inflammation on D3 across all strains were linearly related to necrosis. Our results suggest that (1) dysferlin is not required for functional recovery 3 days after LSI; (2) B6.A/J mice recover from LSI erratically; (3) SJL/J and B10.SJL muscles recover rapidly, perhaps due to ongoing myopathy; (4) although they recover function to different levels, all 4 dysferlinopathic strains show increased inflammation and necrosis 3 days after LSI.

Unmasking potential intracellular roles for dysferlin through improved immunolabeling methods.

Mutations in the DYSF gene that severely reduce the levels of the protein dysferlin are implicated in muscle-wasting syndromes known as dysferlinopathies. Although studies of its function in skeletal muscle have focused on its potential role in repairing the plasma membrane, dysferlin has also been found, albeit inconsistently, in the sarcoplasm of muscle fibers. The aim of this article is to study the localization of dysferlin in skeletal muscle through optimized immunolabeling methods. We studied the localization of dysferlin in control rat skeletal muscle using several different methods of tissue collection and subsequent immunolabeling. We then applied our optimized immunolabeling methods on human cadaveric muscle, control and dystrophic human muscle biopsies, and control and dysferlin-deficient mouse muscle. Our data suggest that dysferlin is present in a reticulum of the sarcoplasm, similar but not identical to those containing the dihydropyridine receptors and distinct from the distribution of the sarcolemmal protein dystrophin. Our data illustrate the importance of tissue fixation and antigen unmasking for proper immunolocalization of dysferlin. They suggest that dysferlin has an important function in the internal membrane systems of skeletal muscle, involved in calcium homeostasis and excitation-contraction coupling.

Physiological and histological changes in skeletal muscle following in vivo gene transfer by electroporation.

Electroporation (EP) is used to transfect skeletal muscle fibers in vivo, but its effects on the structure and function of skeletal muscle tissue have not yet been documented in detail. We studied the changes in contractile function and histology after EP and the influence of the individual steps involved to determine the mechanism of recovery, the extent of myofiber damage, and the efficiency of expression of a green fluorescent protein (GFP) transgene in the tibialis anterior (TA) muscle of adult male C57Bl/6J mice. Immediately after EP, contractile torque decreased by ∼80% from pre-EP levels. Within 3 h, torque recovered to ∼50% but stayed low until day 3. Functional recovery progressed slowly and was complete at day 28. In muscles that were depleted of satellite cells by X-irradiation, torque remained low after day 3, suggesting that myogenesis is necessary for complete recovery. In unirradiated muscle, myogenic activity after EP was confirmed by an increase in fibers with central nuclei or developmental myosin. Damage after EP was confirmed by the presence of necrotic myofibers infiltrated by CD68+ macrophages, which persisted in electroporated muscle for 42 days. Expression of GFP was detected at day 3 after EP and peaked on day 7, with ∼25% of fibers transfected. The number of fibers expressing green fluorescent protein (GFP), the distribution of GFP+ fibers, and the intensity of fluorescence in GFP+ fibers were highly variable. After intramuscular injection alone, or application of the electroporating current without injection, torque decreased by ∼20% and ∼70%, respectively, but secondary damage at D3 and later was minimal. We conclude that EP of murine TA muscles produces variable and modest levels of transgene expression, causes myofiber damage due to the interaction of intramuscular injection with the permeabilizing current, and that full recovery requires myogenesis.

Extensive mononuclear infiltration and myogenesis characterize recovery of dysferlin-null skeletal muscle from contraction-induced injuries.

We studied the response of dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice.

Induced focal adhesion kinase expression suppresses apoptosis by activating NF-kappaB signaling in intestinal epithelial cells.

Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-kappaB signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-kappaB activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF-alpha plus cycloheximide (TNF-alpha/CHX). Specific inhibition of NF-kappaB by the recombinant adenoviral vector containing the IkappaBalpha superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-kappaB activity and increased the sensitivity to TNF-alpha/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-kappaB activity and resulted in increased resistance to TNF-alpha/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-alpha/CHX-induced apoptosis, at least partially, through the activation of NF-kappaB signaling in IECs.