I am looking at peritoneal macrophages and neutrophils in mice that have extreme peritonitis. The non-survivors have decreased phagocytosis (pHrodo e.coli) and reactive oxygen burst (DHR-123, stim with FcR or PMA) as compared to survivors.
How could I determine whether the decrease is due to immuno-suppression or due to the cells being spent from the "battle"?
You can use bioparticles (flourescently labeled bacteria) to feed them and then test them to determine the extend of phagocytosis using flow cytometry. You can also use unlabeled bacteria and H2O2 to determine the oxidative burst also by flow cytometry. Immunosuppression effect may be tested by correlating your findings with cortisol levels of your mice.
Fagoaga, OR (O R)
Latest papers:
Int J Obes Relat Metab Disord. 2002 Jun ;26 (6):822-9 12037653 Cit:6 Influence of skinfold sum and peak VO(2) on immune function in children.
[My paper] D C Nieman, D A Henson, O R Fagoaga, S L Nehlsen-Cannarella, G Sonnenfeld, A C Utter
Department of Health, Leisure, and Exercise Science, Appalachian State University, Boone, North Carolina 28608, USA. [email protected]
OBJECTIVE To measure the relationship of skinfold sum and peak VO(2) power with immune function in children. DESIGN Cross-sectional, with all children tested twice during a 2 month period for peak VO(2), sum of two skinfolds, and immune function, with data from the two measures averaged and then correlated (alpha level,< or = 0.01). Immune measures included leukocyte and lymphocyte subset counts, delayed-typed hypersensitivity (DTH), global IgG antibody response over 4 weeks to pneumococcal vaccination (pIgG), salivary IgA concentration (sIgA), PHA-stimulated lymphocyte proliferation (PHA-SLP), natural killer cell activity (NKCA), and granulocyte and monocyte phagocytosis and oxidative burst activity. SUBJECTS Seventy-three children (n=42 males, n=31 females) ranging in age from 7 to 13 y (mean+/-s.d. age, 9.9+/-1.7 y). The mean skinfold sum was 28.9+/-17.1 mm, and peak VO(2) 45.8+/-8.1 ml/kg/min. RESULTS Peak VO(2), skinfold sum, and immune measures did not differ significantly by age or gender. Therefore, correlations were made on combined indices for all subjects. Peak VO(2) and the skinfold sum were not significantly correlated with NKCA, oxidative burst activity, plgG or DTH. Peak VO(2) was negatively correlated with monocyte phagocytosis (r=-0.30, P=0.012) and positively correlated with PHA-SLP (6.25 microg/ml; r=0.35, P=0.004). The skinfold sum was positively correlated with the total leukocyte count (r=0.39, P4 wk to induce testes regression. Blood was drawn from the ocular sinus or splenocytes obtained to assess basal indexes of immune cell function. In hamsters in short days, natural killer cell cytolytic capacity, as well as spontaneous blastogenesis in both whole blood and isolated lymphocytes, were enhanced compared with that in hamsters in long days. By contrast, phagocytosis and oxidative burst activity by both granulocytes and monocytes were suppressed in hamsters by exposure to short days versus long days. Selective changes in immune cell function coincided with short-day-induced gonadal atrophy. These findings raise the hypothesis that photoperiod regulation of physiological adaptations, including distinct immune cell functions, may help individuals anticipate seasonal challenges posed by opportunistic diseases or climate to facilitate survival. Med Sci Sports Exerc. 1995 Jul ;27 (7):986-92 7564985 Cit:46 Immune function in marathon runners versus sedentary controls.
[My paper] D C Nieman, K S Buckley, D A Henson, B J Warren, J Suttles, J C Ahle, S Simandle, O R Fagoaga, S L Nehlsen-Cannarella
Department of Health, Leisure, and Exercise Science, Applachian State University, Boone, NC, USA.
Marathon runners (N = 22) who had completed at least seven marathons (X +/- SEM = 23.6 +/- 5.7) and had been training for marathon race events for at least 4 yr (12.3 +/- 1.3) were compared with sedentary controls (N = 18). Although the two groups were of similar age (38.7 +/- 1.5 and 43.9 +/- 2.2 yr, respectively) and height, the marathon runners were significantly leaner and possessed a VO2max 60% higher than that of the controls. Neutrophil counts tended to be lower in the group of marathoners, while other leukocyte and lymphocyte subsets were similar to controls. Mitogen-induced lymphocyte proliferation did not differ between groups. Natural killer cell cytotoxic activity (NKCA) was significantly higher in the marathoners versus controls (373 +/- 38 vs 237 +/- 41 total lytic units, respectively, a 57% difference, P = 0.02). For all subjects combined (N = 40) and within the group of marathon runners (N = 22), percent body fat was negatively correlated with NKCA (r =-0.48, P = 0.002; r =-0.49, P = 0.019, respectively), and age was negatively correlated with Con A-induced lymphocyte proliferation (r =-0.41, P = 0.009; r =-0.53, P = 0.011, respectively). These data indicate that NKCA but not mitogen-induced lymphocyte proliferation is higher in marathon runners relative to sedentary controls. Am J Clin Nutr. 1997 Jul ;66 (1):153-9 9209184 Cit:44 Carbohydrate supplementation affects blood granulocyte and monocyte trafficking but not function after 2.5 h or running.
[My paper] D C Nieman, O R Fagoaga, D E Butterworth, B J Warren, A Utter, J M Davis, D A Henson, S L Nehlsen-Cannarella
Department of Health, Appalachian State University, Boone, NC 28608, USA. [email protected]
This randomized, double-blind, placebo-controlled study was designed to determine the influence of carbohydrate supplementation on the granulocyte and monocyte response to 2.5 h of high-intensity running [76.7 +/- 0.4% of maximal oxygen consumption (VO2max)]. Thirty experienced marathon runners (VO2max 53.4 +/- 1.0 mL.kg-1.min-1, age 41.5 +/- 1.4 y) were randomly assigned to carbohydrate-supplement (n = 17) and placebo (n = 13) groups. Subjects rested for 10-15 min before a blood sample was taken at 0715, and then ingested 0.75 L carbohydrate beverage or placebo. At 0730 subjects began running at 75-80% of VO2max for 2.5 h, and drank 0.25 L carbohydrate or placebo fluid every 15 min. Immediately after the 2.5-h run (1000), another blood sample was taken, followed by 1.5-h, 3-h, and 6-h recovery samples. Carbohydrate supplementation had a significant effect compared with placebo on the pattern of change in plasma glucose and cortisol, and the blood concentration of neutrophils (F[14, 112]= 5.13, P = 0.001) and monocytes (F[14, 112]= 4.78, P = 0.001), but not on blood granulocyte and monocyte phagocytosis or oxidative burst activity after 2.5 h of intensive running. J Appl Physiol. 1998 Apr ;84 (4):1252-9 9516191 Cit:44 Effects of mode and carbohydrate on the granulocyte and monocyte response to intensive, prolonged exercise.
[My paper] D C Nieman, S L Nehlsen-Cannarella, O R Fagoaga, D A Henson, A Utter, J M Davis, F Williams, D E Butterworth
Departments of Health, Leisure, and Exercise Science and Biology, Appalachian State University, Boone, North Carolina 28608, USA.
The influence of exercise mode and 6% carbohydrate (C) vs. placebo (P) beverage ingestion on granulocyte and monocyte phagocytosis and oxidative burst activity (GMPOB) after prolonged and intensive exertion was measured in 10 triathletes. The triathletes acted as their own controls and ran or cycled for 2.5 h at approximately 75% maximal O2 uptake, ingesting C or P (4 total sessions, random order, with beverages administered in double-blind fashion). During the 2. 5-h exercise bouts, C or P (4 ml/kg) was ingested every 15 min. Five blood samples were collected (15 min before exercise, immediately after exercise, and 1.5, 3, and 6 h after exercise). The pattern of change over time for GMPOB was significantly different between C and P conditions (P
Defects in phagocytosis are commonly found in severe infections/sepsis. There are a variety of mediators which have been demonstrated in various settings including in-vitro and in-vivo models and in patients - interestingly several of these mediators are considered 'pro-inflammatory' e.g. C5a and fMLP, or are released in large quantities during severe infection e.g. catecholamines and prostaglandins. Classic 'anti-inflammatory' mediators such as IL-10 have little if any effect on phagocytosis.
Blocking these mediators can prevent defects in phagocytosis developing in animal models at least. As a result I think the term 'immunosupression', which tends to invoke ideas of anti-inflammatory/counter-regulatory mediators is a less useful idea than 'immunoparesis'.
In terms of rescue therapy, which I suppose would point towards 'immunoparesis' (i.e the cells are rescuable), my group has demonstrated the efficacy of GM-CSF in vitro and are currently working on a clinical trial of this. Rauch et al's 2012 paper in Science identifying a GM-CSF secreting population of B-cells role in preserving neutrophil function in sepsis provides further insight into this area.
Some papers which might be of interest include:
Huber-Lang M et al, J Immunol 2002
Conway Morris A et al, AJRCCM 2009 and Blood 2011
Rauch P et al, Science , 2012
Fraser J et al J Clin Invest 1981
You could add endotoxin to the cell suspension, incubate for 24h and measure the levels of TNF and IL10 produced by the cells. If the cells are suppressed, the TNF production but not IL10 should be attenuated compared to controls. If they are 'exhausted' both cytokines should be attenuated. Search: LPS tolerance, endotoxin tolerance.
@Seppe: I use a viability stain, as well as spinning the peritoneal exudates over high density media (dead cells and RBCs pellet, viable cells float). What I meant by suppression was that the massive inflammatory storm tries to turn itself off, before the infection is resolved, meanwhile the ongoing infection calls in more cells that are shut down too (immuno-paralysis). I stain with gr-1/ly6g,cd11b, cd19/cd3e, cd45, sytox blue, Dhr123, pHrodo, to measure moncytes/PMN, activation, b-cells/t-tells, wbcs, viability, reactive oxygen, and phagocytosis, respectively.
At 24 hours post cecal ligation and puncture(clp), non-survivors have increased macs, neutrophils, and bacterial CFU in the belly. However, at 6 hours post clp, survivors and non-survivors have the same # of macs, neutrophils, and bacteria. This suggests immune phagocyte impairment (already published by our group).
@seppe, can you provide me a reference for immuno-suppresed macrophages phagocytosing more?
@ Andrew: Thank you for those papers! The Gm-csf paper sounds interesting as it seems to support what i've been observing.
@markus- I've tried that already. There are no difference in cytokine secretion when stimulated by LPS, presumably because the cells have been bathed in it for hours prior to culturing.
Great ideas to think about here, but unfortunately I do not have a clearer approach on how to discern between exhausted cells versus suppressed (paresis-ed)
@ seppe: whereas "spent" would mean the cells are exhausted from fighting the infection.
I'm not clear what you mean by 'non-surviviors'. Do you mean peritoneal cells from mice that don't survive the peritonitis?
yes. We use plasma IL-6 to predict survivors/non-survivors. mice with high il-6 succumb within the first 5 days of sepsis onset
Your question of inhibition vs. over-activation is a central problem not only in sepsis but also in burn and trauma research. The issue arises because of the 'bell shaped' response to stimulation of innate immune cells. From a resting position, first, more stimulation results in higher response (more phagocytosis, migration, etc). After certain threshold however, the increased simulation results in decreasing levels of response. One comparable curve response to help understand the 'bell curve' response is the response of an organism to stress: for low levels of stress, attention is enhanced, higher level of stress are damaging and severely impairing attention.
One way to differentiate between inhibition and over-activation is to treat the cells with small amounts of a mild inhibitor . If the function of interest decreases further, the cells must have been inhibited. If the function of interest increases, the cells must have been over-stimulated.
One example for how these principles are applied to the problem of neutrophil migration after burn injuries is our recent work to come out in FASEB: Kurihara et al 2013. (http://www.fasebj.org/content/early/2013/02/21/fj.12-219519.full.pdf for now). The situation is more complex than I described before, because the drug we used is not an inhibitor but rather a homeostasis restoring drug. However, the principle is overall the same. If you have questions, shoot me an email.
I'm not sure what peritonitis model you are using, but if you used GFP expressing bacteria in a slurry you could then see whether the cells had already phagocytosed before your ex vivo phagocytosis challenge.
Thank you all for the ideas to chew on! I look forward to reading the suggested publications.
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