The slow-release adiponectin analog ALY688-SR modifies early-stage disease development in the D2.mdx mouse model of Duchenne muscular dystrophy.

Fibrosis is associated with respiratory and limb muscle atrophy in Duchenne muscular dystrophy (DMD). Current standard of care partially delays the progression of this myopathy but there remains an unmet need to develop additional therapies. Adiponectin receptor agonism has emerged as a possible therapeutic target to lower inflammation and improve metabolism in mdx mouse models of DMD but the degree to which fibrosis and atrophy are prevented remain unknown. Here, we demonstrate that the recently developed slow-release peptidomimetic adiponectin analog, ALY688-SR, remodels the diaphragm of murine model of DMD on DBA background (D2.mdx) mice treated from days 7-28 of age during early stages of disease. ALY688-SR also lowered interleukin-6 (IL-6) mRNA but increased IL-6 and transforming growth factor-ß1 (TGF-ß1) protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also attenuated fibrosis, fiber type-specific atrophy, and in vitro diaphragm force production in diaphragm suggesting a complex relationship between adiponectin receptor activity, muscle remodeling, and force-generating properties during the very early stages of disease progression in murine model of DMD on DBA background (D2.mdx) mice. In tibialis anterior, the modest fibrosis at this young age was not altered by treatment, and atrophy was not apparent at this young age. These results demonstrate that short-term treatment of ALY688-SR in young D2.mdx mice partially prevents fibrosis and fiber type-specific atrophy and lowers force production in the more disease-apparent diaphragm in relation to lower mitochondrial redox stress and heterogeneous responses in certain inflammatory markers. These diverse muscle responses to adiponectin receptor agonism in early stages of DMD serve as a foundation for further mechanistic investigations.NEW & NOTEWORTHY There are limited therapies for the treatment of Duchenne muscular dystrophy. As fibrosis involves an accumulation of collagen that replaces muscle fibers, antifibrotics may help preserve muscle function. We report that the novel adiponectin receptor agonist ALY688-SR prevents fibrosis in the diaphragm of D2.mdx mice with short-term treatment early in disease progression. These responses were related to altered inflammation and mitochondrial functions and serve as a foundation for the development of this class of therapy.

Copper infused fabric attenuates inflammation in macrophages.

While inflammation is an important immune response for protection from infections, excessive or prolonged inflammation can lead to a variety of debilitating diseases including skin disease, diabetes, heart disease, stroke, autoimmune diseases and cancer. Inflammation is a graded response that is typically initiated when resident macrophages sense the presence of pathogens or damage in the tissue and produce inflammatory cytokines and chemokines to kill the pathogen, clear debris and dead tissue, and initiate tissue repair. Here we show that copper-infused fabrics can prevent inflammation by blocking the production of inflammatory cytokines from macrophages after being exposed to LPS, a component of bacterial cell wall. Mechanistically, we show that copper-infused fabrics can significantly reduce the NF-κB and IRF3 activation in LPS-stimulated macrophages. Given the importance of excessive inflammation in diabetes, we show that copper can reduce insulin resistance mediated by inflammatory cytokines in muscle cells. Our data show that copper infused fabrics may be useful to reduce excessive inflammation in macrophages and improve insulin sensitivity in skeletal muscles.

Moderate exercise induces trained immunity in macrophages.

Despite its importance in protecting the host from infections and injury, excessive inflammation may lead to serious human diseases including autoimmune disorders, cardiovascular diseases, diabetes, and cancer. Exercise is a known immunomodulator; however, whether exercise causes long-term changes in inflammatory responses and how these changes occur are lacking. Here, we show that chronic moderate-intensity training of mice leads to persistent metabolic rewiring and changes to chromatin accessibility in bone marrow-derived macrophages (BMDMs), which, in turn, tempers their inflammatory responses. We show that BMDMs from exercised mice exhibited a decrease in lipopolysaccharide (LPS)-induced NF-κB activation and proinflammatory gene expression along with an increase in M2-like-associated genes when compared with BMDMs from sedentary mice. This was associated with improved mitochondrial quality and increased reliance on oxidative phosphorylation accompanied with reduced mitochondrial reactive oxygen species (ROS) production. Mechanistically, assay for transposase-accessible chromatin (ATAC)-seq analysis showed changes in chromatin accessibility of genes associated with inflammatory and metabolic pathways. Overall, our data suggest that chronic moderate exercise can influence the inflammatory responses of macrophages by reprogramming their metabolic and epigenetic landscape.NEW & NOTEWORTHY In this study, we explain how long-term moderate exercise training can reduce inflammation in mouse macrophages by reprogramming the way they sense and respond to the presence of pathogens. We completed a thorough analysis and showed that these changes persist in macrophages because exercise improves the ability of cells to utilize oxygen without producing damaging compounds, and changes the way they access their DNA.

Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells.

Iron overload is associated with various pathological changes which contribute to metabolic syndrome, many of which have been proposed to occur via damaging tissue through an excessive amount of reactive oxygen species (ROS) production. In this study, we established a model of iron overload in L6 skeletal muscle cells and observed that iron enhanced cytochrome c release from depolarized mitochondria, assayed by immunofluorescent colocalization of cytochrome c with Tom20 and the use of JC-1, respectively. This subsequently elevated apoptosis, determined via use of a caspase-3/7 activatable fluorescent probe and western blotting for cleaved caspase-3. Using CellROX deep red and mBBr, we observed that iron increased generation of reactive oxygen species (ROS), and that pretreatment with the superoxide dismutase mimetic MnTBAP reduced ROS production and attenuated iron-induced intrinsic apoptosis and cell death. Furthermore, using MitoSox Red we observed that iron enhanced mROS and the mitochondria-targeted anti-oxidant SKQ1 reduced iron-induced ROS generation and cell death. Western blotting for LC3-II and P62 levels as well as immunofluorescent detection of autophagy flux with LC3B and P62 co-localization indicated that iron acutely (2-8 h) activated and later (12-24 h) attenuated autophagic flux. We used autophagy-deficient cell models generated by overexpressing a dominant-negative Atg5 mutant or CRISPR-mediated ATG7 knock out to test the functional significance of autophagy and observed that autophagy-deficiency exacerbated iron-induced ROS production and apoptosis. In conclusion, our study showed that high iron levels promoted ROS production, blunted the self-protective autophagy response and led to cell death in L6 skeletal muscle cells.

The Acute Effects of Milk Consumption on Systemic Inflammation after Combined Resistance and Plyometric Exercise in Young Adult Females.

High-intensity/impact exercise elicits a transient increase in inflammatory biomarkers. Consuming nutrient-dense wholefoods, like milk, following exercise may modulate post-exercise inflammation and aid recovery. We examined the effect of post-exercise skim milk consumption (versus an isoenergetic, isovolumetric carbohydrate [CHO] drink) on acute exercise-induced inflammation in untrained females. Using a randomized crossover design, 13 healthy females (age = 20 ± 2.3 y; BMI = 21.0 ± 1.1 kg/m2) completed two bouts of combined resistance/plyometric exercise followed by either skim milk (MILK) or CHO at 5-min and 1 h post-exercise. Serum interleukin [IL]-1ß, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α) concentrations were measured at pre-exercise, 15-min, 75-min, 24 h, and 48 h post-exercise. IL-6 increased 15-min post-exercise vs. all other timepoints (time effect, p = 0.017). Between 24 and 48 h, IL-10 decreased and increased in the MILK and CHO conditions, respectively (interaction, p = 0.018). There were no significant effects for IL-1ß or TNF-α. Relative concentrations of IL-1ß (p = 0.049) and IL-10 (p = 0.028) at 48 h post-exercise were lower in MILK vs. CHO. Milk post-exercise did not influence the absolute concentration of pro-inflammatory cytokines; however, there were divergent responses for the anti-inflammatory cytokine, IL-10, and milk reduced the relative inflammatory response at 48 h (vs. CHO) for IL-1ß and IL-10. This demonstrates the potential for milk to modulate inflammation post-exercise in this sample.

Role of Mitochondrial Iron Overload in Mediating Cell Death in H9c2 Cells.

Iron overload (IO) is associated with cardiovascular diseases, including heart failure. Our study's aim was to examine the mechanism by which IO triggers cell death in H9c2 cells. IO caused accumulation of intracellular and mitochondrial iron as shown by the use of iron-binding fluorescent reporters, FerroOrange and MitoFerroFluor. Expression of cytosolic and mitochondrial isoforms of Ferritin was also induced by IO. IO-induced iron accumulation and cellular ROS was rapid and temporally linked. ROS accumulation was detected in the cytosol and mitochondrial compartments with CellROX, DCF-DA and MitoSOX fluorescent dyes and partly reversed by the general antioxidant N-acetyl cysteine or the mitochondrial antioxidant SkQ1. Antioxidants also reduced the downstream activation of apoptosis and lytic cell death quantified by Caspase 3 cleavage/activation, mitochondrial Cytochrome c release, Annexin V/Propidium iodide staining and LDH release of IO-treated cells. Finally, overexpression of MitoNEET, an outer mitochondrial membrane protein involved in the transfer of Fe-S clusters between mitochondrial and cytosol, was observed to lower iron and ROS accumulation in the mitochondria. These alterations were correlated with reduced IO-induced cell death by apoptosis in MitoNEET-overexpressing cells. In conclusion, IO mediates H9c2 cell death by causing mitochondrial iron accumulation and subsequent general and mitochondrial ROS upregulation.

The PKN1- TRAF1 signaling axis as a potential new target for chronic lymphocytic leukemia.

TRAF1 is a pro-survival adaptor molecule in TNFR superfamily (TNFRSF) signaling. TRAF1 is overexpressed in many B cell cancers including refractory chronic lymphocytic leukemia (CLL). Little has been done to assess the role of TRAF1 in human cancer. Here we show that the protein kinase C related kinase Protein Kinase N1 (PKN1) is required to protect TRAF1 from cIAP-mediated degradation during constitutive CD40 signaling in lymphoma. We show that the active phospho-Thr774 form of PKN1 is constitutively expressed in CLL but minimally detected in unstimulated healthy donor B cells. Through a screen of 700 kinase inhibitors, we identified two inhibitors, OTSSP167, and XL-228, that inhibited PKN1 in the nanomolar range and induced dose-dependent loss of TRAF1 in RAJI cells. OTSSP167 or XL-228 treatment of primary patient CLL samples led to a reduction in TRAF1, pNF-κB p65, pS6, pERK, Mcl-1 and Bcl-2 proteins, and induction of activated caspase-3. OTSSP167 synergized with venetoclax in inducing CLL death, correlating with loss of TRAF1, Mcl-1, and Bcl-2. Although correlative, these findings suggest the PKN1-TRAF1 signaling axis as a potential new target for CLL. These findings also suggest the use of the orally available inhibitor OTSSP167 in combination treatment with venetoclax for TRAF1 overexpressing CLL.

Innate immunity drives pathogenesis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease affecting ∼1% of the general population. This disease is characterized by persistent articular inflammation and joint damage driven by the proliferating synovial tissue fibroblasts as well as neutrophil, monocyte and lymphocyte trafficking into the synovium. The factors leading to RA pathogenesis remain poorly elucidated although genetic and environmental factors have been proposed to be the main contributors to RA. The majority of the early studies focused on the role of lymphocytes and adaptive immune responses in RA. However, in the past two decades, emerging studies showed that the innate immune system plays a critical role in the onset and progression of RA pathogenesis. Various innate immune cells including monocytes, macrophages and dendritic cells are involved in inflammatory responses seen in RA patients as well as in driving the activation of the adaptive immune system, which plays a major role in the later stages of the disease. Here we focus the discussion on the role of different innate immune cells and components in initiation and progression of RA. New therapeutic approaches targeting different inflammatory pathways and innate immune cells will be highlighted here. Recent emergence and the significant roles of innate lymphoid cells and inflammasomes will be also discussed.

Dicer regulates activation of the NLRP3 inflammasome.

Inflammation plays a critical role in initiation of adaptive immunity, pathogen clearance and tissue repair. Interleukin (IL)-1ß is a potent pro-inflammatory cytokine and therefore its production is tightly regulated: its secretion requires the assembly of a macromolecular protein complex, termed the inflammasome. Aberrant activation of the inflammasome has been linked to debilitating human diseases including chronic inflammatory and autoimmune diseases. Thus, there is a great interest in understanding how inflammasomes are regulated. Here we show that Dicer, an enzyme necessary for the production of mature micro-RNAs (miRNAs), is required for optimal activation of NLRP3 inflammasomes in bone marrow macrophages. Our data indicate that miRNAs may play an important role in promoting inflammasome activation.

The Evolving Role of TRAFs in Mediating Inflammatory Responses.

TRAFs [tumor necrosis factor (TNF) receptor associated factors] are a family of signaling molecules that function downstream of multiple receptor signaling pathways and play a pivotal role in the biology of innate, and adaptive immune cells. Following receptor ligation, TRAFs generally function as adapter proteins to mediate the activation of intracellular signaling cascades. With the exception of TRAF1 that lacks a Ring domain, TRAFs have an E3 ubiquitin ligase activity which also contributes to their ability to activate downstream signaling pathways. TRAF-mediated signaling pathways culminate in the activation of several transcription factors, including nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs; e.g., ERK-1 and ERK-2, JNK, and p38), and interferon-regulatory factors (IRF; e.g., IRF3 and IRF7). The biological role of TRAFs is largely due to their ability to positively or negatively regulate canonical and non-canonical NF-κB signaling. While TRAF-mediated signaling regulates various immune cell functions, this review is focused on the recent advances in our knowledge regarding the molecular mechanisms through which TRAF proteins regulate, positively and negatively, inflammatory signaling pathways, including Toll-IL-1 receptors, RIG-I like receptors, and Nod-like receptors. The review also offers a perspective on the unanswered questions that need to be addressed to fully understand how TRAFs regulate inflammation.

TRAF1 Signaling in Human Health and Disease.

Tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) is a signaling adaptor first identified as part of the TNFR2 signaling complex. TRAF1 plays a key role in pro-survival signaling downstream of TNFR superfamily members such as TNFR2, LMP1, 4-1BB, and CD40. Recent studies have uncovered another role for TRAF1, independent of its role in TNFR superfamily signaling, in negatively regulating Toll-like receptor and Nod-like receptor signaling, through sequestering the linear ubiquitin assembly complex, LUBAC. TRAF1 has diverse roles in human disease. TRAF1 is overexpressed in many B cell related cancers and single nucleotide polymorphisms (SNPs) in TRAF1 have been linked to non-Hodgkin's lymphoma. Genome wide association studies have identified an association between SNPs in the 5' untranslated region of the TRAF1 gene with increased incidence and severity of rheumatoid arthritis and other rheumatic diseases. The loss of TRAF1 from chronically stimulated CD8 T cells results in desensitization of the 4-1BB signaling pathway, thereby contributing to T cell exhaustion during chronic infection. These apparently opposing roles of TRAF1 as both a positive and negative regulator of immune signaling have led to some confusion in the literature. Here we review the role of TRAF1 as a positive and negative regulator in different signaling pathways. Then we discuss the role of TRAF1 in human disease, attempting to reconcile seemingly contradictory roles based on current knowledge of TRAF1 signaling and biology. We also discuss avenues for future research to further clarify the impact of TRAF1 in human disease.

Dichotomous Expression of TNF Superfamily Ligands on Antigen-Presenting Cells Controls Post-priming Anti-viral CD4+ T Cell Immunity.

T cell antigen-presenting cell (APC) interactions early during chronic viral infection are crucial for determining viral set point and disease outcome, but how and when different APC subtypes contribute to these outcomes is unclear. The TNF receptor superfamily (TNFRSF) member GITR is important for CD4+ T cell accumulation and control of chronic lymphocytic choriomeningitis virus (LCMV). We found that type I interferon (IFN-I) induced TNFSF ligands GITRL, 4-1BBL, OX40L, and CD70 predominantly on monocyte-derived APCs and CD80 and CD86 predominantly on classical dendritic cells (cDCs). Mice with hypofunctional GITRL in Lyz2+ cells had decreased LCMV-specific CD4+ T cell accumulation and increased viral load. GITR signals in CD4+ T cells occurred after priming to upregulate OX40, CD25, and chemokine receptor CX3CR1. Thus IFN-I (signal 3) induced a post-priming checkpoint (signal 4) for CD4+ T cell accumulation, revealing a division of labor between cDCs and monocyte-derived APCs in regulating T cell expansion.

The signaling adaptor TRAF1 negatively regulates Toll-like receptor signaling and this underlies its role in rheumatic disease.

TRAF1 is a signaling adaptor known for its role in tumor necrosis factor receptor-induced cell survival. Here we show that monocytes from healthy human subjects with a rheumatoid arthritis-associated single-nucleotide polymorphism (SNP) in the TRAF1 gene express less TRAF1 protein but greater amounts of inflammatory cytokines in response to lipopolysaccharide (LPS). The TRAF1 MATH domain binds directly to three components of the linear ubiquitination (LUBAC) complex, SHARPIN, HOIP and HOIL-1, to interfere with the recruitment and linear ubiquitination of NEMO. This results in decreased NF-κB activation and cytokine production, independently of tumor necrosis factor. Consistent with this, Traf1-/- mice show increased susceptibility to LPS-induced septic shock. These findings reveal an unexpected role for TRAF1 in negatively regulating Toll-like receptor signaling, providing a mechanistic explanation for the increased inflammation seen with a disease-associated TRAF1 SNP.

Different STAT Transcription Complexes Drive Early and Delayed Responses to Type I IFNs.

IFNs, which transduce pivotal signals through Stat1 and Stat2, effectively suppress the replication of Legionella pneumophila in primary murine macrophages. Although the ability of IFN-γ to impede L. pneumophila growth is fully dependent on Stat1, IFN-αß unexpectedly suppresses L. pneumophila growth in both Stat1- and Stat2-deficient macrophages. New studies demonstrating that the robust response to IFN-αß is lost in Stat1-Stat2 double-knockout macrophages suggest that Stat1 and Stat2 are functionally redundant in their ability to direct an innate response toward L. pneumophila. Because the ability of IFN-αß to signal through Stat1-dependent complexes (i.e., Stat1-Stat1 and Stat1-Stat2 dimers) has been well characterized, the current studies focus on how Stat2 is able to direct a potent response to IFN-αß in the absence of Stat1. These studies reveal that IFN-αß is able to drive the formation of a Stat2 and IFN regulatory factor 9 complex that drives the expression of a subset of IFN-stimulated genes, but with substantially delayed kinetics. These observations raise the possibility that this pathway evolved in response to microbes that have devised strategies to subvert Stat1-dependent responses.

Cyclic-di-GMP and cyclic-di-AMP activate the NLRP3 inflammasome.

The cyclic dinucleotides 3'-5'diadenylate (c-diAMP) and 3'-5' diguanylate (c-diGMP) are important bacterial second messengers that have recently been shown to stimulate the secretion of type I Interferons (IFN-Is) through the c-diGMP-binding protein MPYS/STING. Here, we show that physiologically relevant levels of cyclic dinucleotides also stimulate a robust secretion of IL-1ß through the NLRP3 inflammasome. Intriguingly, this response is independent of MPYS/STING. Consistent with most NLRP3 inflammasome activators, the response to c-diGMP is dependent on the mobilization of potassium and calcium ions. However, in contrast to other NLRP3 inflammasome activators, this response is not associated with significant changes in mitochondrial potential or the generation of mitochondrial reactive oxygen species. Thus, cyclic dinucleotides activate the NLRP3 inflammasome through a unique pathway that could have evolved to detect pervasive bacterial pathogen-associated molecular patterns associated with intracellular infections.

Reversible inhibition of Chlamydia trachomatis infection in epithelial cells due to stimulation of P2X(4) receptors.

Bacterial infections of the mucosal epithelium are a major cause of human disease. The prolonged presence of microbial pathogens stimulates inflammation of the local tissues, which leads to changes in the molecular composition of the extracellular milieu. A well-characterized molecule that is released to the extracellular milieu by stressed or infected cells is extracellular ATP and its ecto-enzymatic degradation products, which function as signaling molecules through ligation of purinergic receptors. There has been little information, however, on the effects of the extracellular metabolites on bacterial growth in inflamed tissues. Millimolar concentrations of ATP have been previously shown to inhibit irreversibly bacterial infection through ligation of P2X(7) receptors. We show here that the proinflammatory mediator, ATP, is released from Chlamydia trachomatis-infected epithelial cells. Moreover, further stimulation of the infected cells with micromolar extracellular ADP or ATP significantly impairs the growth of the bacteria, with a profile characteristic of the involvement of P2X(4) receptors. A specific role for P2X(4) was confirmed using cells overexpressing P2X(4). The chlamydiae remain viable and return to normal growth kinetics after removal of the extracellular stimulus, similar to responses previously described for persistence of chlamydial infection.

The overlapping host responses to bacterial cyclic dinucleotides.

Macrophages respond to infection with Legionella pneumophila by the induction of inflammatory mediators, including type I Interferons (IFN-Is). To explore whether the bacterial second messenger cyclic 3'-5' diguanylate (c-diGMP) activates some of these mediators, macrophages were infected with L. pneumophila strains in which the levels of bacterial c-diGMP had been altered. Intriguingly, there was a positive correlation between c-diGMP levels and IFN-I expression. Subsequent studies with synthetic derivatives of c-diGMP, and newly described cyclic 3'-5' diadenylate (c-diAMP), determined that these molecules activate overlapping inflammatory responses in human and murine macrophages. Moreover, UV crosslinking studies determined that both dinucleotides physically associate with a shared set of host proteins. Fractionation of macrophage extracts on a biotin-c-diGMP affinity matrix led to the identification of a set of candidate host binding proteins. These studies suggest that mammalian macrophages can sense and mount a specific inflammatory response to bacterial dinucleotides.

Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection.

Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches.

Inflammasome-dependent caspase-1 activation in cervical epithelial cells stimulates growth of the intracellular pathogen Chlamydia trachomatis.

Inflammasomes have been extensively characterized in monocytes and macrophages, but not in epithelial cells, which are the preferred host cells for many pathogens. Here we show that cervical epithelial cells express a functional inflammasome. Infection of the cells by Chlamydia trachomatis leads to activation of caspase-1, through a process requiring the NOD-like receptor family member NLRP3 and the inflammasome adaptor protein ASC. Secretion of newly synthesized virulence proteins from the chlamydial vacuole through a type III secretion apparatus results in efflux of K(+) through glibenclamide-sensitive K(+) channels, which in turn stimulates production of reactive oxygen species. Elevated levels of reactive oxygen species are responsible for NLRP3-dependent caspase-1 activation in the infected cells. In monocytes and macrophages, caspase-1 is involved in processing and secretion of pro-inflammatory cytokines such as interleukin-1beta. However, in epithelial cells, which are not known to secrete large quantities of interleukin-1beta, caspase-1 has been shown previously to enhance lipid metabolism. Here we show that, in cervical epithelial cells, caspase-1 activation is required for optimal growth of the intracellular chlamydiae.