Inhibitory effect of polyunsaturated fatty acids on apoptosis induced by Streptococcus pneumonia in alveolar macrophages

Background & objectives:  Apoptosis is considered as a major defense mechanism of the body.   Multiple pathogens induce macrophage apoptosis as a mode of immune evasion.  In earlier studies, n3  polyunsaturated fatty acids (PUFA) have been reported to be protective against neuronal apoptosis and neuronal degeneration, seen after spinal cord injury.  In this study, we tried to evaluate the role of n3  polyunsaturated fatty acids on the process of macrophage phagocytic activity and apoptosis in mice.
Methods:  Mice were divided into three groups (n of 60); Group I was fed on sea cod oil; Group II on flaxseed oil supplementation for 9 wk along with standard laboratory chow diet.  Group III was fed on standard diet and served as control.  After supplementation, phagocytic and apoptotic (morphological staining:  acridine orange plus ethidium bromide; H-33342 plus propidium iodide staining and DNA ladder formation) activities of mouse alveolar macrophages were assessed. Results:  Alveolar macrophages (obtained from sea cod oil and flaxseed oil fed group mice) showed significant increase in bacterial uptake as well as intracellular killing (P<0.05) of Streptococcus pneumonia.  Significant decrease (P<0.05) in apoptotic cells was observed among alveolar macrophages from sea cod and flaxseed oil fed mice whereas maximum apoptosis was observed in control alveolar macrophages on interaction with bacteria in vitro which was confirmed by DNA laddering. Interpretation & conclusions:  These findings suggest that dietary supplementation with n3  polyunsatrated fatty acids to mice led to enhanced phagocytic capability of their alveolar macrophages as well as provided protection against apoptosis upon challenge with S.pneumoniae. (Authors abstract)

Pneumococcus usually colonizes the nasopharynx of humans asymptomatically, although sometimes it moves to the lungs, brain, and blood.  Pneumococci are capable of inducing apoptosis in respiratory tree epithelium, endothelium, neuronal cells and alveolar macrophages (AM).    AM are the first time defenders in the lungs and play an essential role against infections because of their capability to phagocytose and kill the invading microorganisms.  To induce and potentiate inflammatory immune processes, AM release the required cellular mediators. Oxidative stress activates apoptosis, and antioxidants protect against apoptosis in vitro; thus, a central role of dietary antioxidants may be to protect against apoptosis.  Omega 3 polyunsaturated fatty acids are important nutritional elements for humans, and these have the potential to inhibit excessive inflammatory responses; hence are widely recommended against atherosclerosis, coronary heart diseases, arrhythmias and in allergic conditions like asthma.  In an attempt to understand the role of dietary n3  polyunsaturated fatty acids (PUFA) on phagocytosis and apoptosis, the interaction of alveolar macrophages obtained from the mouse fed on n3  PUFA with Streptococcus pneumonia, in vitro was assessed. 

In the present study, phagocytosis of S. pneumonia with mouse alveolar macrophages obtained from animals supplemented with n3  PUFA for 9 wks was studied.  When compared with control, macrophages showed significant increase in uptake as well as intracellular killing (P<0.05) of S. pneumonia.  Macrophages from sea cod oil fed group showed 30 per cent increase in phagocytic uptake which was slightly higher than the flaxseed oil group (27%).  Similarly, increase in intracellular killing of S. pneumonia by alveolar macrophages from sea cod oil as well as flaxseed oil fed groups was 36 and 30 per cent, respectively.  Previous studies have observed that macrophages enriched with saturated fatty acids such as myristate or palmitate showed decrease of 28 and 21 per cent, respectively in their ability to phagocytose unopsonized zymosan particles.  Those enriched with polyunsaturated fatty acids showed 25 to 55 per cent enhancement of phagocytic capacity.  A difference in H-33342 fluorescence emission between normal and apoptotic cells is attributed to a more rapid membrane uptake of H-33342 by cells undergoing apoptosis.  The potential of S. pneumonia to induce apoptosis in alveolar macrophages of n3  PUFA supplemented mice was evaluated.  In order to quantify the apoptotic population, the nuclear morphology of cells was observed following acridine orange plus ethidium bromide staining and H-33342 plus propidium iodide staining.  The macrophages in the control samples were moderate green.  The apoptotic cells were greenish yellow in colour whereas the necrotic cells were orange-red.  With H-33342 plus propidium iodide staining viable cells showed moderate blue fluorescence while apoptic cells showed nuclei with bright pink.  Significant decrease in apoptotic cells was observed among alveolar macrophages from sea cod and flaxseed oil fed mice whereas maximum apoptosis was observed in control alveolar macrophages on interaction with bacteria in vitro which was confirmed by DNA laddering.

The present study demonstrated that dietary supplementation with n3  PUFA not only enhanced phagocytic capability of alveolar macrophages but also decreased alveolar macrophage apoptosis by S. pneumonia D39 type 2.  Modulation of macrophage life span can be an important mechanism for the regulation of macrophage function.  The incorporation of n3  PUFA in immune cell membranes may influence the membrane fluidity, structure and function of several membrane receptors, transporters, enzymes and ionic channels.  These alterations can in turn indirectly modulate macrophage function as has been reported in earlier studies related to central nervous system associated problems. In conclusion, the present observations are important as AM are the first line of defence in respiratory tract infections.  These observations, therefore, from the basis for future experimentation to study the molecular basis of altered macrophage function on exposure to n3  PUFA. (Editors comments)