Inflammatory cytokine responses to progressive resistance training and supplementation with fortified milk in men aged 50+ years : an 18-month randomized controlled trial

We examined the effects of progressive resistance training (PRT) and supplementation with calcium-vitamin D(3) fortified milk on markers of systemic inflammation, and the relationship between inflammation and changes in muscle mass, size and strength. Healthy men aged 50-79 years (n = 180) participated in this 18-month randomized controlled trial that comprised a factorial 2 x 2 design. Participants were randomized to (1) PRT + fortified milk supplement, (2) PRT, (3) fortified milk supplement, or (4) a control group. Participants assigned to PRT trained 3 days per week, while those in the supplement groups consumed 400 ml day(-1) of milk containing 1,000 mg calcium plus 800 IU vitamin D(3). We collected venous blood samples at baseline, 12 and 18 months to measure the serum concentrations of IL-6, TNF-alpha and hs-CRP. There were no exercise x supplement interactions, but serum IL-6 was 29% lower (95% CI, -62, 0) in the PRT group compared with the control group after 12 months. Conversely, IL-6 was 31% higher (95% CI, -2, 65) in the supplement group compared with the non-supplemented groups after 12 and 18 months. These between-group differences did not persist after adjusting for changes in fat mass. In the PRT group, mid-tibia muscle cross-sectional area increased less in men with higher pre-training inflammation compared with those men with lower inflammation (net difference similar to 2.5%, p < 0.05). In conclusion, serum IL-6 concentration decreased following PRT, whereas it increased after supplementation with fortified milk concomitant with changes in fat mass. Furthermore, low-grade inflammation at baseline restricted muscle hypertrophy following PRT.

Characterization of inflammatory responses to eccentric exercise in humans

Eccentric exercise commonly results in muscle damage. The primary sequence of events leading to exercise-induced muscle damage is believed to involve initial mechanical disruption of sarcomeres, followed by impaired excitation-contraction coupling and calcium signaling, and finally, activation of calcium-sensitive degradation pathways. Muscle damage is characterized by ultrastructural changes to muscle architecture, increased muscle proteins and enzymes in the bloodstream, loss of muscular strength and range of motion and muscle soreness. The inflammatory response to exercise-induced muscle damage is characterized by leukocyte infiltration and production of pro-inflammatory cytokines within damaged muscle tissue, systemic release of leukocytes and cytokines, in addition to alterations in leukocyte receptor expression and functional activity. Current evidence suggests that inflammatory responses to muscle damage are dependent on the type of eccentric exercise, previous eccentric loading (repeated bouts), age and gender. Circulating neutrophil counts and systemic cytokine responses are greater after eccentric exercise using a large muscle mass (e.g. downhill running, eccentric cycling) than after other types of eccentric exercise involving a smaller muscle mass. After an initial bout of eccentric exercise, circulating leukocyte counts and cell surface receptor expression are attenuated. Leukocyte and cytokine responses to eccentric exercise are impaired in elderly individuals, while cellular infiltration into skeletal muscle is greater in human females than males after eccentric exercise. Whether alterations in intracellular calcium homeostasis influence inflammatory responses to muscle damage is uncertain. Furthermore, the effects of antioxidant supplements are variable, and the limited data available indicates that anti-inflammatory drugs largely have no influence on inflammatory responses to eccentric exercise. In this review, we compare local versus systemic inflammatory responses, and discuss some of the possible mechanisms regulating the inflammatory responses to exercise-induced muscle damage in humans.

Changes in inflammatory mediators following eccentric exercise of the elbow flexors

The aims of this study were to examine the plasma concentrations of inflammatory mediators including cytokines induced by a single bout of eccentric exercise and again 4 weeks later by a second bout of eccentric exercise of the same muscle group. Ten untrained male subjects performed two bouts of the eccentric exercise involving the elbow flexors (6 sets of 5 repetitions) separated by four weeks. Changes in muscle soreness, swelling, and function following exercise were compared between the bouts. Blood was sampled before, immediately after, 1 h, 3 h, 6 h, 24 h (1 d), 48 h (2 d), 72 h (3 d), 96 h (4 d) following exercise bout to measure plasma creatine kinase (CK) activity, plasma concentrations of myoglobin (Mb), interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1ra), IL-4, IL-6, IL-8, IL-10, IL-12p40, tumor necrosis factor (TNF)-alpha, granulocyte colony-stimulating factor (G-CSF), myeloperoxidase (MPO), prostaglandin E2 (PGE2), heat shock protein (HSP) 60 and 70. After the first bout, muscle soreness increased significantly, and there was also significant increase in upper arm circumference; muscle function decreased and plasma CK activity and Mb concentration increased significantly. These changes were significantly smaller after the second bout compared to the first bout, indicating muscle adaptation to the repeated bouts of the eccentric exercise. Despite the evidence of greater muscle damage after the first bout, the changes in cytokines and other inflammatory mediators were quite minor, and considerably smaller than that following endurance exercise. These results suggest that eccentric exercise-induced muscle damage is not associated with the significant release of cytokines into the systemic circulation. After the first bout, plasma G-CSF concentration showed a small but significant increase, whereas TNF-alpha and IL-8 showed significant decreases compared to the pre-exercise values. After the second bout, there was a significant increase in IL-10, and a significant decrease in IL-8. In conclusion, although there was evidence of severe muscle damage after the eccentric exercise, this muscle damage was not accompanied by any large changes in plasma cytokine concentrations. The minor changes in systemic cytokine concentration found in this study might reflect more rapid clearance from the circulation, or a lack of any significant metabolic or oxidative demands during this particular mode of exercise. In relation to the adaptation to the muscle damage, the anti-inflammatory cytokine IL-10 might work as one of the underlying mechanisms of action.

Intracellular trafficking and secretion of inflammatory cytokines

The secretion of cytokines by immune cells plays a significant role in determining the course of an inflammatory response. The levels and timing of each cytokine released are critical for mounting an effective but confined response, whereas excessive or dysregulated inflammation contributes to many diseases. Cytokines are both culprits and targets for effective treatments in some diseases. The multiple points and mechanisms that have evolved for cellular control of cytokine secretion highlight the potency of these mediators and the fine tuning required to manage inflammation. Cytokine production in cells is regulated by cell signaling, and at mRNA and protein synthesis levels. Thereafter, the intracellular transport pathways and molecular trafficking machinery have intricate and essential roles in dictating the release and activity of cytokines. The trafficking machinery and secretory (exocytic) pathways are complex and highly regulated in many cells, involving specialized membranes, molecules and organelles that enable these cells to deliver cytokines to often-distinct areas of the cell surface, in a timely manner. This review provides an overview of secretory pathways - both conventional and unconventional - and key families of trafficking machinery. The prevailing knowledge about the trafficking and secretion of a number of individual cytokines is also summarized. In conclusion, we present emerging concepts about the functional plasticity of secretory pathways and their modulation for controlling cytokines and inflammation.

Pro-inflammatory growth factors reduce secondary degeneration after traumatic spinal cord injury

The role of inflammatory response after spinal cord injury remains unclear. This thesis was a step forward in studying how promoting the inflammation, by delivery pro-inflammatory growth factors, affects the outcomes of spinal cord injury. A significant functional improvement was observed after treatment and these results suggest an interesting avenue for future clinical treatments and may provide a platform to improve the efficacy of other treatments.

Repeated intrauterine exposures to inflammatory stimuli attenuated transforming growth factor-β signaling in the ovine fetal lung

Background: Bronchopulmonary dysplasia (BPD) is one of the most common complications after preterm birth and is associated with intrauterine exposure to bacteria. Transforming growth factor-β (TGFβ) is implicated in the development of BPD. Objectives: We hypothesized that different and/or multiple bacterial signals could elicit divergent TGFβ signaling responses in the developing lung. Methods: Time-mated pregnant Merino ewes received an intra-amniotic injection of lipopolysaccharide (LPS) and/or Ureaplasma parvum serovar 3 (UP) at 117 days' and/or 121/122 days' gestational age (GA). Controls received an equivalent injection of saline and or media. Lambs were euthanized at 124 days' GA (term = 150 days' GA). TGFβ1, TGFβ2, TGFβ3, TGFβ receptor (R)1 and TGFβR2 protein levels, Smad2 phosphorylation and elastin deposition were evaluated in lung tissue. Results: Total TGFβ1 and TGFβ2 decreased by 24 and 51% after combined UP+LPS exposure, whereas total TGFβ1 increased by 31% after 7 days' LPS exposure but not after double exposures. Alveolar expression of TGFβR2 decreased 75% after UP, but remained unaltered after double exposures. Decreased focal elastin deposition after single LPS exposure was prevented by double exposures. Conclusions: TGFβ signaling components and elastin responded differently to intrauterine LPS and UP exposure. Multiple bacterial exposures attenuated TGFβ signaling and normalized elastin deposition.

Results of a phase IIa clinical trial of an anti-inflammatory molecule, chaperonin 10, in multiple sclerosis

Background Chaperonin 10 (Cpn10) is a mitochondrial molecule involved in protein folding. The aim of this study was to determine the safety profile of Cpn10 in patients with multiple sclerosis (MS). Methods A total of 50 patients with relapse-remitting or secondary progressive MS were intravenously administered 5 mg or 10 mg of Cpn10 weekly for 12 weeks in a double-blind, randomized, placebo controlled, phase II trial. Clinical reviews, including Expanded Disability Status Scale and magnetic resonance imaging (MRI) with Gadolinium, were undertaken every 4 weeks. Stimulation of patient peripheral blood mononuclear cells with lipopolysaccharide ex vivo was used to measure the in vivo activity of Cpn10. Results No significant differences in the frequency of adverse events were seen between treatment and placebo arms. Leukocytes from both groups of Cpn10-treated patients produced significantly lower levels of critical proinflammatory cytokines. A trend toward improvement in new Gadolinium enhancing lesions on MRI was observed, but this difference was not statistically significant. No differences in clinical outcome measures were seen. Conclusions Cpn10 is safe and well tolerated when administered to patients with MS for 3 months, however, a further extended phase II study primarily focused on efficacy is warranted.

Common PPARγ variants C161T and Pro12Ala are not associated with inflammatory bowel disease in an Australian cohort

Background & Aims: Peroxisome proliferator-activated receptor (PPAR) γ is a transcription factor, highly expressed in colonic epithelial cells, adipose tissue and macrophages, with an important role in the regulation of inflammatory pathways. The common PPARγ variants C161T and Pro12Ala have recently been associated with Ulcerative Colitis (UC) and an extensive UC phenotype respectively, in a Chinese population. PPARγ Pro12Ala variant homozygotes appear to be protected from the development of Crohn's disease (CD) in European Caucasians. Methods: A case-control study was performed for both variants (CD n=575, UC n=306, Controls n=360) using a polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis in an Australian IBD cohort. A transmission disequilibrium test was also performed using CD trios for the PPARγ C161T variant. Genotype-phenotype analyses were also undertaken. Results: There was no significant difference in genotype distribution data or allele frequency between CD and UC patients and controls. There was no difference in allele transmission for the C161T variant. No significant relationship between the variants and disease location was observed. Conclusions: We were unable to replicate in a Caucasian cohort the recent association between PPARγ C161T and UC or between PPARγ Pro12Ala and an extensive UC phenotype in a Chinese population. There are significant ethnic differences in genetic susceptibility to IBD and its phenotypic expression.

Packed red blood cells suppress myeloid dendritic cell and monocyte inflammatory responses in a whole blood model of transfusion

Purpose/Objective: The basis for poor outcomes in some patients post transfusion remains largely unknown. Despite leukodepletion, there is still evidence of immunomodulatory effects of transfusion that require further study. In addition, there is evidence that the age of blood components transfused significantly affects patient outcomes. Myeloid dendritic cell (DC) and monocyte immune function were studied utilising an in vitro whole blood model of transfusion. Materials and methods: Freshly collected (‘recipient’) whole blood was cultured with ABO compatible leukodepleted PRBC at 25% blood replacement-volume (6hrs). PRBC were assayed at [Day (D) 2, 14, 28and 42 (date-of expiry)]. In parallel, LPS or Zymosan (Zy) were added to mimic infection. Recipients were maintained for the duration of the time course (2 recipients, 4 PRBC units, n = 8).Recipient DC and monocyte intracellular cytokines and chemokines (IL-6, IL-10, IL-12,TNF-a, IL-1a, IL-8, IP-10, MIP-1a, MIP-1b, MCP-1) were measured using flow cytometry. Changes in immune response were calculated by comparison to a parallel no transfusion control (Wilcoxin matched pairs). Influence of storage age was calculated using ANOVA. Results: Significant suppression of DC and monocyte inflammatory responses were evident. DC and monocyte production of IL-1a was reduced following exposure to PRBC regardless of storage age (P < 0.05 at all time points). Storage independent PRBC mediated suppression of DC and monocyte IL-1a was also evident in cultures costimulated with Zy. In cultures co-stimulated with either LPS or Zy, significant suppression of DC and monocyte TNF-a and IL-6 was also evident. PRBC storage attenuated monocyte TNF-a production when co-cultured with LPS (P < 0.01 ANOVA). DC and monocyte production of MIP-1a was significantly reduced following exposure to PRBC (DC: P < 0.05 at D2, 28, 42; Monocyte P < 0.05 all time points). In cultures co-stimulated with LPS and zymosan, a similar suppression of MIP-1a production was also evident, and production of both DC and monocyte MIP-1b and IP-10 were also significantly reduced. Conclusions: The complexity of the transfusion context was reflected in the whole blood approach utilised. Significant suppression of these key DC and monocyte immune responses may contribute to patient outcomes, such as increased risk of infection and longer hospital stay, following blood transfusion.

Bridging the biology of the ovine TRALI model – human inflammatory cell responses to TRALI inducing supernatants

Background Transfusion-related acute lung injury (TRALI) is a serious and potentially fatal consequence of transfusion. A two-event TRALI model demonstrated date-of-expiry - day (D) 5 platelet (PLT) and D42 packed red blood cell (PRBC) supernatants (SN) induced TRALI in LPS-treated sheep. We have adapted a whole blood transfusion culture model as an investigative bridge between the ovine TRALI model human responses to transfusion. Methods A whole blood transfusion model was adapted to replicate the ovine model - specifically +/- 0.23μg/mL LPS as the first event and 10% SN volume (transfusion) as the second event. Four pooled SN from blood products, previously used in the TRALI ovine model, were investigated: D1-PLT, D5-PLT, D1-PRBC, and D42-PRBC. Fresh human whole blood (recipient) was mixed with combinations of LPS and BP-SN stimuli and incubated in vitro for 6 hrs. Addition of golgi plug enabled measurement of monocyte cytokine production (IL-6, IL-8, IL-10, IL-12, TNF-α, IL-1α, CXCL-5, IP-10, MIP-1α, MCP-1) using multi-colour flow cytometry. Responses for 6 recipients were assessed. Results In the presence of LPS, D42-PRBC-SN significantly increased monocyte IL-6 (P=0.031), IL-8 (P=0.016) and IL-1α (P=0.008) production compared to D1-PRBC-SN. This response to D42-PRBC-SN was LPS-dependent, and was not evident in non-LPSstimulated controls. This response was also specific to D42-PRBC-SN, as similar changes were not evident for the D5-PLT-SN, compared to the D1-PLT-SN, regardless of the presence of LPS. D5-PLT-SN significantly increased IL-12 production (P=0.024) compared to D1-PLT-SN. This response was again LPS-dependent. Conclusions These data demonstrate a novel two-event mechanism of monocyte inflammatory response that was dependent upon both the presence of date-of-expiry blood product SN and LPS. Further, these results demonstrate different cytokines responses induced by date-of-expiry PLT-SN and PRBC-SN. These data are consistent with the evidence from the ovine TRALI model, and enhancing its relevance to transfusion related changes in humans.

Mathematical investigation of the interactions between the inflammatory response and mechanical aspects of dermal wound repair

Dermal wound repair involves complex interactions between cells, cytokines and mechanics to close injuries to the skin. In particular, we investigate the contribution of fibroblasts, myofibroblasts, TGFβ, collagen and local tissue mechanics to wound repair in the human dermis. We develop a morphoelastic model where a realistic representation of tissue mechanics is key, and a fibrocontractive model that involves a reasonable approximation to the true kinetics of the important bioactive species. We use each of these descriptions to elucidate the mechanisms that generate pathologies such as hypertrophic scars, contractures and keloids. We find that for hypertrophic scar and contracture development, factors regulating the myofibroblast phenotype are critical, with heightened myofibroblast activation, reduced myofibroblast apoptosis or prolonged inflammation all predicted as mediators for scar hypertrophy and contractures. Prevention of these pathologies is predicted when myofibroblast apoptosis is induced, myofibroblast activation is blocked or TGFβ is neutralised. To investigate keloid invasion, we develop a caricature representation of the fibrocontractive model and find that TGFβ spread is the driving factor behind keloid growth. Blocking activation of TGFβ is found to cause keloid regression. Thus, we recommend myofibroblasts and TGFβ as targets for clinicians when developing intervention strategies for prevention and cure of fibrotic scars.

Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses : implications for laminitis

Equine laminitis, a disease of the lamellar structure of the horse’s hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Tolllike receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-�), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-� and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-�, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease.

Clinical dyslipidaemia is associated with changes in the lipid composition and inflammatory properties of apolipoprotein-B-containing lipoproteins from women with type 2 diabetes

The aim of this study was to use lipidomics to determine if the lipid composition of apolipoprotein-B-containing lipoproteins is modified by dyslipidaemia in type 2 diabetes and if any of the identified changes potentially have biological relevance in the pathophysiology of type 2 diabetes. VLDL and LDL from normolipidaemic and dyslipidaemic type 2 diabetic women and controls were isolated and quantified with HPLC and mass spectrometry. A detailed molecular characterisation of VLDL triacylglycerols (TAG) was also performed using the novel ozone-induced dissociation method, which allowed us to distinguish vaccenic acid (C18:1 n-7) from oleic acid (C18:1 n-9) in specific TAG species. Lipid class composition was very similar in VLDL and LDL from normolipidaemic type 2 diabetic and control participants. By contrast, dyslipidaemia was associated with significant changes in both lipid classes (e.g. increased diacylglycerols) and lipid species (e.g. increased C16:1 and C20:3 in phosphatidylcholine and cholesteryl ester and increased C16:0 [palmitic acid] and vaccenic acid in TAG). Levels of palmitic acid in VLDL and LDL TAG correlated with insulin resistance, and VLDL TAG enriched in palmitic acid promoted increased secretion of proinflammatory mediators from human smooth muscle cells. We showed that dyslipidaemia is associated with major changes in both lipid class and lipid species composition in VLDL and LDL from women with type 2 diabetes. In addition, we identified specific molecular lipid species that both correlate with clinical variables and are proinflammatory. Our study thus shows the potential of advanced lipidomic methods to further understand the pathophysiology of type 2 diabetes.