Role of toll-like receptor 4 in skeletal muscle damage in chronic limb-threatening ischemia

Objective Toll-like receptors (TLRs) are key pattern recognition receptors in the innate immune system. In particular, the TLR4-mediated immune response has been implicated in ischemia-induced tissue injury. Mounting evidence supports a detrimental role of the innate immune system in the pathophysiology of skeletal muscle damage in patients with chronic limb-threatening ischemia (CLTI), in whom patient-oriented functional outcomes are poor. The overall aim of this study was to investigate the potential role of TLR4 in skeletal muscle dysfunction and damage in CLTI. Methods The role of TLR4 in ischemic muscle was investigated by (1) studying TLR4 expression and distribution in human gastrocnemius muscle biopsies, (2) evaluating the functional consequences of TLR4 inhibition in myotubes derived from human muscle biopsies, and (3) assessing the therapeutic potential of modulating TLR4 signaling in ischemic muscle in a mouse hindlimb ischemia model. Results TLR4 was found to be expressed in human muscle biopsies, with significant upregulation in samples from patients with CLTI. In vitro studies using cultured human myotubes demonstrated upregulation of TLR4 in ischemia, with activation of the downstream signaling pathway. Inhibition of TLR4 before ischemia was associated with reduced ischemia-induced apoptosis. Upregulation of TLR4 also occurred in ischemia in vivo and TLR4 inhibition was associated with decreased inflammatory cell infiltration and diminished apoptosis in the ischemic limb. Conclusions TLR4 is upregulated and activated in ischemic skeletal muscle in patients with CLTI. Modulating TLR4 signaling in vitro and in vivo was associated with attenuation of ischemia-induced skeletal muscle damage. This strategy could be explored further for potential clinical application.

The prevalence of peripheral arterial disease (PAD) is growing and is a global health care burden with important public health implications. 1,2Although #38% of these patients are asymptomatic, the rest experience some degree of symptoms ranging from mild intermittent claudication to the most severe form that is chronic limb-threatening ischemia (CLTI).Successful revascularization in patients with CLTI contributes to limb salvage; however, this process does not equate to a return to premorbid ambulatory or occupational status. 3 This finding suggests that adjunctive treatments in addition to revascularization to improve both clinical and patient-focused outcomes should be explored.5][6] Skeletal muscle forms the bulk of the lower limb and plays a key part in its function.Further, skeletal muscle has a role as a paracrine signaling organ, whereby pro-inflammatory cytokines released from the damaged muscle can trigger a systemic inflammatory response.
The innate immune system recognizes damageassociated molecular patterns, upregulated in any tissue damage, via pattern recognition receptors.Among the better-characterized pattern recognition receptors are the Toll-like receptors (TLRs).The immune response mediated by TLRs is protective in most cases.However, if inflammation becomes chronic, excessive tissue damage may occur, contributing to the pathogenesis of diseases such as atherosclerosis. 7,8Recognition of damage-associated molecular patterns by TLRs results in the recruitment of adaptor proteins and activation of downstream signaling pathways: the MyD88dependent and TRIF-dependent pathways.Each pathway starts the activation of specific transcription factors such as nuclear factor kB (NF-kB), Jun N-terminal kinase (JNK), and mitogen-activated protein kinases, which are required for inflammatory gene transcription.This in turn results in the release of a wide range of cytokines and inflammatory markers such as IL1, IL6, IL8, tumor necrosis factor-a (TNF-a), interferon-a and interferon-b. 9LR4 has a pathogen detection role and acts as a monitoring receptor in the detection of tissue injury.The release of TLR4 endogenous ligands is associated with TLR4 activation and the corresponding inflammatory response can lead to excessive tissue damage.Exogenous inhibition of TLR4 in an animal model of myocardial ischemia has been associated with decreased ischemic injury and inflammation. 10he overall aim of this study was to investigate the potential role of TLR4 in skeletal muscle dysfunction in CLTI.The role of TLR4 in ischemic muscle was studied with three aims: (1) to explore TLR4 expression and distribution in muscle biopsies from patients with CLTI, (2) to investigate the TLR4 pathway and functional consequences of TLR4 inhibition in myotubes derived from human muscle biopsies, and (3) to evaluate the therapeutic potential of modulating TLR4 signaling in ischemic muscle using a relevant mousehind limb ischemia model.

METHODS
Gastrocnemius biopsies were taken from the medial head of the gastrocnemius muscle from patients undergoing major lower limb amputation for CLTI (ischemic group) and from patients with no PAD undergoing saphenous vein harvesting for coronary artery bypass surgery (control group).Western blot analysis and immunostaining were carried out to study TLR4 expression and distribution, and to explore the downstream signaling pathways.
Further, human myoblasts were isolated, cultured to myotubes, and then exposed to simulated ischemia.The isolated myoblasts were examined every 48 hours to monitor for 80% confluence before induction to differentiate into myotubes.Enzyme-linked immunosorbent assay (ELISA), Western blot, and immunostaining were used to study the role of TLR4 signaling pathway inhibition in ischemia-induced apoptosis and cytokine release from myotubes.To study the therapeutic potential of modulating TLR4 signaling in ischemic skeletal muscle, a mouse model of hindlimb ischemia was used.The femoral artery was ligated with a sterile 7-0 Prolene suture at the level of the inguinal ligament proximally and just above the popliteal artery distally.All the side branches were ligated with 7-0 Prolene suture.Laser Doppler and immunostaining were carried out to assess the effect of TLR4 inhibition on skeletal muscle damage in vivo.For in vivo studies, animals were sacrificed (n ¼ 6 per ischemic groups and n ¼ 3 per sham groups at each time point).This number was chosen based on published studies of TLR changes in ischemia as well as studies employing similar analyses in mouse hindlimb ischemia models. 10,11JMP 11.0.0 software (SAS Institute, Cary, NC) was used to present, describe, and analyze the data from this study.Further information on the methods and materials in this study is detailed in the Supplemental Materials.
All mice showed low functional performance after surgery with gradual improvement by day 21.Mice in TLR4 À/À and LPS-RS groups restored their functional status to near baseline by day 21, whereas the control group had lower performance even at day 21 (P < .05;Kruskal-Wallis test) (Fig 5).The blood gas analyses of blood samples taken via cardiac puncture at the time of sacrificing the animals showed no significant differences between the ischemic and sham-operated mice in either the control or TLR4 À/À groups when the following parameters were quantified: pH, pO 2 , pCO 2 , hemoglobin, lactate, Na, K, and SaO 2 .The random blood glucose levels in the ischemic and sham-operated TLR4 À/À groups were significantly higher compared with ischemic and shamoperated control groups at days À1, 3, 7, and 21 (P < .05;Kruskal-Wallis test).The ELISA quantification of IL-6 showed significantly increased levels in the control group compared with TLR4 À/À and LPS-RS groups at days 3, 7, and 21 after surgery (P < .05;Kruskal-Wallis test).Similarly, ELISA quantification of TNF-a showed significantly increased levels in the control group compared with TLR4 À/À and LPS-RS groups at days 3, 7, and 21 after surgery (P < .05;Kruskal-Wallis test).
Fluorescent immunohistochemical staining for cleaved-caspase 3 showed less expression of this protein in TLR4-inhibited groups at day 21 (Fig 6).Inflammatory cell quantification, carried out using ImageJ 1.4s (US National Institutes of Health) in hematoxylin and eosinstained, paraffin-embedded ischemic gastrocnemius muscle sections after surgery showed less inflammatory cell infiltration in both TLR4 À/À and LPS-RS groups when compared with the control group at each time point (P < .05;Kruskal-Wallis test) (Fig 6 .)The median percentages of inflammatory cell infiltration in the control, TLR4 À/À , and LPS-RS groups were calculated as 19.0%, 7.0%, and 10.5%, respectively, at day 3; 21.0%, 10.0%, and 14.0%, respectively, at day 7; and 14.0%, 8.0%, and 10.5%, respectively, at day 21.TLR4 À/À mice and mice given LPS-RS demonstrated attenuated histological evidence of ischemia-induced inflammation after hindlimb ischemia as compared with control mice at days 3, 7, and 21.The hematoxylin and eosin examination of the hindlimb skeletal muscles in sham groups showed no obvious histological differences between the control, TLR4 À/À , and LPS-RS groups at days 3, 7, and 21.

DISCUSSION
Understanding the pathophysiology of the ischemiainduced inflammatory damage in CLTI may provide a potential missing link in improving functional outcomes along with successful revascularization.It is thought that the muscle dysfunction in CLTI is potentially due to ischemia-induced muscle damage, in which inflammation plays a central role. 5,12e showed that TLR4 is expressed in both ischemic and nonischemic human skeletal muscle, with increased expression in the ischemic samples.TLR4-mediated NF-kB and JNK pathways have been shown to be involved in myocardium ischaemia. 13,14Here, P-NF-kB and pJNK, activated forms of NF-kB and JNK, were significantly upregulated in the ischemic samples.The high expression of these transcription proteins suggests that TLR4 is not only upregulated, but also activated.Further, the increase in cleaved caspase-3, a marker for apoptosis, suggests that significant tissue damage occurs even within clinically viable lower limb muscle.This finding supports previous studies implicating TLR-induced apoptotic cell death in the pathophysiology of ischemia-induced cell damage. 15Immunofluorescence staining of the muscle biopsies revealed the presence of TLR4 on neutrophils, endothelium, and macrophages.
In vitro experiments showed that exposure of mature myotubes to nutrition depletion, hypoxia, and hypercapnia resulted in a significant time-dependent increase in the percentage of apoptotic cells and inflammatory cytokine release, which are features of skeletal muscle ischemia in vivo.Simulated ischemia in cultured human myotubes also led to the upregulation of TLR4 and activation of its downstream signaling pathway with increased cytokine release and apoptosis in the ischemic human myotubes.Further, inhibition of TLR4 before ischemia was associated with inhibition of the signaling pathway and reduced ischemia-induced apoptosis.Moreover, amplified cytokine release and increased expression of HSP60 and HSP70 were demonstrated in ischemic skeletal muscle in vitro.Inhibition of the The ischemic/nonischemic lower limb perfusion ratio showed a significant difference on days 3, 7, and 21 between the TLR4 À/À group and control and TLR4 antagonist (LPS-RS) groups (*P < .001at days 3 and 7; **P ¼ .01 at day 21; Kruskal-Wallis test).The difference between each group was also significant (P < .005;Mann-Whitney U test) on days 3, 7, and 21.RS-LPS, lipopolysaccharide from Rhodobacter sphaeroides.
MyD88-dependent signaling pathway attenuated inflammatory cytokine production in the presence of ischemiainduced TLR4 activation.These data are in keeping with previous findings of accelerated muscle regeneration in hindlimb ischemia in MyD88 knockout mice, 16 and supports the involvement of MyD88 in the inflammatory response to ischemia, which may be a critical step in the development of skeletal muscle damage.Furthermore, TLR4 antagonism was associated with reduced inflammatory cytokine release and downregulation of HSP60 and HSP70 expressions.This finding suggests a potential pathway where TLR4 and its endogenous ligands contribute to a positive feedback loop to maintain a proinflammatory environment during ischemia.The in vivo experiments showed that TLR4 is upregulated in ischemic skeletal muscle.TLR4 À/À mice and mice given LPS-RS, a TLR4 antagonist, exhibited decreased systemic IL6 and TNF-a levels after hindlimb ischemia, implicating the role of TLR4 in ischemia-induced systemic inflammatory cytokine production.Both endogenous and exogenous inhibition of TLR4 were associated with decreased inflammatory cell infiltration and diminished apoptosis in the ischemic limb as compared with the control group.
The overall limitation of our study is the need for a more in-depth analysis of the signaling pathways and mechanisms involved, because the crosstalk between the different signaling pathways can affect the final response.Further studies are required to explore different drug delivery routes and timing and the doseresponse effect of TLR4 antagonists.Although TLR antagonists have been shown clearly to be protective by directly inhibiting inflammatory gene transcription and decreasing the secretion of inflammatory cytokines in several conditions, preconditioning with low-dose TLR agonists may also be an effective strategy to protect tissue against subsequent detrimental insults such as reperfusion injury.This result highlights the importance of administering specific TLR agonists and antagonists during the relevant phase of the pathological process to achieve an optimal balance of TLR activation and inhibition. 17This study also has specific limitations regarding the in vivo model: a chronic model of ischemia with gradual occlusion was not performed in our experiments, mainly because we aimed to develop a costeffective model that can be used in most laboratories; we did not evaluate older mice, which may demonstrate more similar responses to our CLTI patients, again owing to practical husbandry and cost considerations.
The TLR4-induced inflammatory response has been shown to encourage angiogenesis and collateral artery formation in various ischemia/reperfusion models, suggesting the dual role of TLR4 in inflammation and angiogenesis.However, it has been reasoned that the fine-tuning of TLR4 and its associated signaling pathway can eliminate the undesired effect of TLR4 inhibition on collateral artery formation. 18van den Borne et al 19 studied the effect of systemic inhibition of TLR4 on perfusion recovery in a mouse model for angiogenesis.They found that inhibition of TLR4 by its specific inhibitor TAK-242 in a mouse model of hindlimb ischemia did not negatively influence perfusion recovery after ischemia, despite its potential inhibitory effects on angiogenesis. 19Moreover, TLR4 can interplay with other inflammatory signaling pathways to balance the final response to ischemia.Inflammation is involved in both muscle regeneration and angiogenesis; however, a regulated balance is required to prevent unnecessary inflammation-induced tissue damage, while preserving the positive effects.It has been reported that unopposed TLR4 activation in skeletal muscle ischemia slows regeneration and also angiogenesis, whereas TLR2, which is simultaneously upregulated by TLR4, plays a protective role in modifying the TLR4 effect. 20Additionally, the other components of the adaptive immune system such as T-cell-mediated responses are further potential areas of future research to explore the possible interactions with the TLR4 signaling pathway.The inhibition of TLR4 can also be considered in adjunct with other novel strategies.Decreasing the ischemia-induced inflammatory response may improve   The potential therapeutic role of TLR4 antagonists should be considered as adjuvant therapy in PAD.Muscle changes including inflammation are documented in patients with intermittent claudication.We envisage a potential therapeutic role of TLR4 antagonists as an adjuvant therapy where earlier intervention before severe muscle damage, alongside revascularization may offer more benefit than treating patients with late-stage CLTI.The risk-benefit ratio would need to be determined based on any side-effects of treatment at each stage.
In summary, this study investigated the role of TLR4mediated inflammatory responses in ischemic skeletal muscle.TLR4 was found to be upregulated and activated in ischemic skeletal muscle in patients with CLTI, and TLR4 signaling contributed to ischemia-induced inflammation and cell/tissue damage in vitro and in vivo ( Fig 7).Modulating TLR4 signaling in vivo was associated with attenuation of skeletal muscle damage.This work highlights the potential therapeutic role of TLR4 inhibition in patients with CLTI.It is conceivable that pharmacological targeting of TLR4 in patients with CLTI together with improving hemodynamics may improve both functional and clinical outcomes.JVSeVascular Science

Fig 5 .
Fig 5. Functional and ischemic scoring of the mice in all groups showed faster recovery in Toll-like receptor (TLR4) À/ À and lipopolysaccharide (LPS-RS) groups compared with the vehicle group (*P < .05 between groups; Kruskal-Wallis test; n ¼ 6).
the environment/niche to enable growth factors or stem cells to function better.

Fig 7 .
Fig 7. Schematic showing the potential link between ischemia-induced Toll-like receptor 4 (TLR4) activation and the resulting tissue damage.Ischemia leads to muscle damage with the release of inflammatory cytokines.Upon activation of TLR4, adaptor proteins (MyD88 and TRIF) and transcription proteins (P38, JNK, and nuclear factor kB [NF-kB]) are recruited to activate the inflammatory gene transcription.

Table I .
Demographics of the two patient groups ABPI, ankle-brachial pressure index; CAD, coronary artery disease; CLTI, chronic limb-threatening ischemia; eGFR, estimated glomerular filtration rate; HTN, hypertension; N/A, not applicable; PAD, peripheral arterial disease.Values are numbers unless otherwise noted.Boldface entries indicate statistical significance.The two groups differed only in terms of the presence of PAD symptoms and reduced ABPI, where P < .05,Chi-square test