Nutritional immunotherapy: An overview

In previous posts (1,2), I have briefly reviewed the importance of adipocytes and molecules secreted by the adipose tissue in immunity. Overall, increases in adiposity alterate the inflammatory balance, shifting towards a pro-inflammatory state, which contributes to the development of obesity-associated diseases. 

Given that metabolic and immune pathways are interconnected, and that metabolism controls function in immune cells, it is possible to modulate the immune system through nutrition. This is what I call "Nutritional Immunotherapy", which simply means targeting the immune system with nutritional tools for treating inflammatory and autoimmune diseases. 

Nutrition acts both directly and indirectly on the immune system, as shown in the following diagram:

Relationship between nutrition and the immune system. See text for details.

1. Nutrition influences the composition and function of adipose tissue (AT): Energy intake regulates fat mass, affecting the function and differentiation of pre- and mature adipocytes (direct effect). The concentration of specific fatty acids in AT is proportional to their abundance in the diet (indirect effect). The dietary fatty acid profile also affects membrane lipid composition on other cell types, which regulates cell functioning.  
2. Adipose tissue is an immune organ: There are several immune cells present in AT from different body sites, differing each one in the proportion of cell types. Lymph nodes present in AT are sorrounded by perinodal adipocytes, which have a higher proportion of polyunsaturated fatty acids (PUFA) than adipocytes far from the nodes.  Lymphoid clusters within AT include milky spots (MS) and fat-associated lymphoid clusters (FALCs), which have an active role determining whole-body immune responses. Adipocytes are also able to secrete adipocytokines (leptin, resistin, adiponectin, etc.) and classical cytokines (IL-6, TNF-a, etc.). 
3. Adipose tissue regulates energy intake: Cytokines secreted by AT regulate appetite and energy balance, acting through neural pathways involved in energy homeostasis.
4. Nutrition affects the gut flora: Microbial composition of the human gut flora is very responsive to diet. Small changes in either macronutrient distribution or food choices affect differently not only the relative proportion of certain species, but also their metabolism and gene expression patterns.
5. Gut flora regulates fat mass and metabolism: Digestion of plant cell walls, oligosaccharides and other food components by gut bacteria increases the energy yield of food, contributing to energy intake. Acetate and propionate, produced by the fermentation of soluble fiber, are metabolized (predominantly) in skeletal muscle and the liver, respectively. Gut microbiota also supress FIAF activity and promotes hepatic triglyceride synthesis. Metabolism of drugs and xenobiotics is dependent on the composition of the gut flora. 
6. Gut flora regulates immunity: The presence of specific bacteria shapes the immune system and regulates mucosal and peripheral immune responses. The gut flora also competes with enteropathogens directly and by the action of antimicrobial peptides. Evolutionary co-adaptation has given gut bacteria and other microorganisms essential roles for mammalian health. 
7. Nutrition regulates immunity: Energy availability and macronutrients regulate the function, maturation and differentiation of immune cells. 

Nutrition has the potential to act on all levels mentioned above. This is why a good diet is very important not only for prevention, but also for treatment of diseases of civilization.

Important components

The nutritional immunotherapy protocol integrates concepts from immunology, molecular and evolutionary biology. The first two help us answer the "how" question, while the latter helps us understand the "why". 

What differences this protocol from other diets is that it takes into account the fact that people who already have developed an inflammatory and/or autoimmune disorder respond differently to any diet. This means that the response to a diet is individual, and more importantly, in this case, the starting point is not a natural one. This point is important for understanding the recommendations given hereafter.  

The history of the patient, specially those aspects that would compromise the response to certain macronutrients and the normal development of a tolerant immune system, needs to be addressed before trying to make any nutritional adjustment. Nowadays, with genetic tools (like 23andMe), tailoring the diet according to the genotype is possible and helpful. 

Important factors for the success and application of the protocol are shown below.

Relevant factors for the protocol

Mode of birth

Hygiene practices during childhood

Family diet, diet history and maternal environment

Medical history

Genotype

Social/lifestyle experiences

Symptoms

Self-assessment

Bloodwork

 Mode of birth

This important but commonly overlooked factor is determinant for immune development and future health. Vaginal birth is the natural mode of birth because it stimulates not only hormonal responses in the mother and the child, but because it promotes an adequate colonization of the neonate, one that we have been adapted for. At birth, the newborn is sterile*, so it can be colonized virtually by any species. Babies born by cesarean section have an abnormal microbiota, as they harbor bacteria from the hospital's environment, medical practitioners and the mother's skin. Normally, during the passage through the birth canal, the infant is exposed to vaginal and cervical flora. Because of its proximity, newborns are also rapidly colonized by maternal gut microbiota, which seems to be the predominant source of bacteria. Pre-term infants also display a different pattern of microbial colonization.

Hygiene practices during childhood 

Gut development is a continuous process that has its last phase during late infancy/early childhood, as the child transitions from breastmilk to complementary foods. Exclusive breastfeeding (and ingestion of colostrum) is very important for preventing inadequate colonization, as it has bacteria, immune and growth factors which promote immune development. Breastmilk also has a perfect nutritional composition, with oligosaccharides (and other components) that promote the growth and establishment of commensal bacteria (predominately Bifidobacteria). Formula-fed infants display an aberrant gut microbiota and normal colonization is severly delayed (if not completely disrupted).

Microbial exposure favors diversification and exposure to pathogens, which is necessary for stimulation of immune memory and tolerance. Contact with animals, eating raw food and playing in the dirt are a necessary part of a healthy lifestyle in infancy. Excessive hygiene and antibiotic use promote dysbiosis. 

Family diet, diet history and maternal environment 

The diet eaten by your father, mother and grandparents influences the expression of genes involved in energy metabolism. These effects are transmitted intergenerationally and lasting during adulthood. Inadequate dietary patterns followed during childhood and adulthood worsen immune and metabolic function. Additionally, maternal status during pregnancy (stress, nutrition, etc.) has profound effects on many genes. 

Medical history 

Previous diseases, antibiotic abuse and utilization of other substances can influence both the normal functioning of the immune system as well as metabolism.

Genotype 

The presence of certain alleles are important for tolerance of specific food components (ie. lactose) and variability in immune responses (ie. MHC alleles, cytokine gene polymorphism).

Social/lifestyle experiences 

Having bad social relationships, lack of optimism, stress and other common lifestyle experiences affect the inflammatory status of the body. For example, losing a game in very competitive persons increases the levels of inflammatory cytokines higher than in non-competitive subjects. Mental stress also seem to affect the composition of the gut microbiota and gut permeability. 

Symptoms, self-assessment and bloodwork

Any symptom (either bad or good) is valuable for trying to identify potential problems. Self-assessment, including anthropometric measures, emotional status or the characteristics of feces can also help narrowing the spectrum of possible disorders. Bloodwork and biomarkers are helpful for confirming assumptions and health status.

Summary

Before beginning any nutritional therapy, it is important to check for past events and factors that affect the metabolic and immune status. This will aid in finding the right dietary composition that helps the most with a given problem and reducing the time of experimentation needed for finding the adequate nutrition and supplementation for an individual.  

*Although recent evidence suggests that bacteria colonize the gut in uterus.

Human microbiota and atherosclerosis

I've been wanting to post about this study for a while now. I think its a good update while I finish my first post on my nutritional immunotherapy protocol. This study was performed given the preliminary evidence linking infections and atherosclerosis, and the association of the human microbiota with the atherosclerotic plaque. For example, bacterial DNA has been observed in atherosclerotic plaques from young and old subjects (1, 2). This relationship has been investigated with more focus on oral bacteria, due to the association of periodontal disease and cardiovascular disease (CVD) (3, 4) and the presence of periodontal pathogens in  atherosclerotic plaques (5). 

The authors tried to answer the following questions:

Is there a core atherosclerotic plaque microbiota? 

Are bacteria present in the plaque also detectable in the oral cavities or guts of the same individuals?

Do the microbiotas of the oral cavity, gut, and atherosclerotic plaque relate to disease markers such as plasma levels of apolipoproteins and cholesterol? 

Is an altered oral or fecal microbiota associated with atherosclerosis?

Using 16S rRNA sequences (from patients with clinical atherosclerosis and controls) and the unweighted UniFrac distance metric (qualitative instead of quantitative), they found strong clustering of samples according to body site, suggesting that the oral, gut and atherosclerotic plaque (AP) sites have different microbial communities:

PC1 and PC2 refer to the first two principal coordinates from the principal coordinate analysis of unweighted UniFrac, plotted for each sample (See also Fig S1). Of these sites, bacterial diversity was higher for the gut microbiota. 

The analysis of the atherosclerotic plaque microbiota revealed that there was a positive correlation between the amount of bacterial 16S rRNA and the number of leukocytes present in the AP, and there was significantly higher levels of Proteobacteria and fewer Firmicutes compared with the oral and gut samples. Supporting the role for a "core" AP microbiota, several OTUs were present in all AP samples, which differentiated these samples from oral or fecal samples: Chryseomonas was detected at high levels in the AP samples, but not in gut or oral samples, being the most discriminative genus between sites and driving the differences between body sites. Other OTUs, three for the genus Staphylococcus, three classified as Propionibacterineae and one belonging to the genus Burkholderia, were specific for AP samples and were present in all AP samples analyzed.

There were no OTUs differentiating oral samples from healthy subjects and patients, but there were correlations between the abundances of OTUs in the oral cavity and CVD markers: the abundance of Fusobacterium was positively correlated with levels of cholesterol (P = 0.028) and LDL (P = 0.005), the abundance of Streptococcus was positively  correlated to HDL (P = 0.0001) and ApoAI (P = 0.01) levels and the abundance of Neisseria was negatively correlated to levels of these last two markers (P = 0.02 and 0.005, respectively). This is interesting, given that Fusobacterium has been associated with periodontal disease (6). As with oral samples, there were no differentiating OTUs between gut samples from controls and patients (in terms of OTU abundances). In gut samples, the abundance of two OTUs classified as uncharacterized members of Erysipelotrichaceae and Lachnospiraceae families were positively correlated with cholesterol (P = 0.009 and 0.001, respectively) and LDL (P = 0.012 and 0.007, respectively). 

Finally, inter-individual comparisons between sites showed that some OTUs were shared among sites. These included OTUs for Veillonella (in AP and oral samples in 11 of 13 patients, detected also in the gut sample of two patients) and Streptococcus (in AP and oral samples in 6 of 10 patients, detected also in the gut of four patients). Within patients, the AP samples contained OTUs shared with oral (Propionibacterium, Rothia, Burkholderia, Corynebacterium, Granulicatella, Staphylococcus) and gut (Bacteroides, Bryantella, Enterobacter, Ruminococcus) samples. 

Summary

• The study identified a "core" atherosclerotic plaque microbiota, comprising higher levels of Proteobacteria and fewer Firmicutes, compared with the gut and oral samples. 
• The AP microbiota contained specific OTUs not shared with the analyzed body sites.
• The abundance of some OTUs in the gut and oral cavity was correlated with CVD markers. 
• Shared OTUs among sites included Streptococcus and Veillonella, the correlation being stronger among the oral cavity and the AP, and these OTUs were also found in the gut samples from some patients. Across patients, the abundance of both were correlated in the oral cavity and the AP.

Commentary

I find this study very interesting because it supports the role of infection on the pathogenesis of atherosclerosis and CVD. The "infection hypothesis" of atherosclerosis has been proposed before (7). The fact that specific bacteria is present in AP and not in other body sites analyzed and that the amount of bacterial 16S rRNA was positively correlated with leukocyte counts, support the notion that these pathogens support directly atherosclerosis progression. However, the study only analyzed the oral cavity and the gut, so it is impossible to conclude that these pathogens couldnt have been derived from other body sites (for example, the skin). Moreover, primers commonly utilized to amplify 16S rRNA sequences are limited to some species, an inherent property of the method (8). Nevertheless, it seems more feasible to suppose that the origin of AP bacteria is the oral cavity because of the close proximity of the bacterial communities in the mouth to the highly vascularized gingival lining and because of the thickness of the subgingival epithelium, which differs from other protective layers such as the skin or the gut mucosa (9). Accordingly, any mechanical disruption of oral bacterial biofilms can trigger bacteremia, and these include oral procedures (periodontal probing, tooth extractions, etc), oral hygiene activities (such as brushing) and physiological phenomena (like chewing) (9). This, coupled with the findings that the abundance of OTUs in the AP were correlated with that of the oral cavity support this hypothesis. Gut bacterial origin is more complicated but feasible (as shown by the presence of gut bacteria in AP samples). The authors suggest that one possible way of this transfer is by phagocytosis of macrophages at epithelial linings. 

If indeed bacteria play a role in the formation and/or progression of atherosclerosis, the million dollar question is why do these specific pathogens adhere to the vascular endothelium? Moreover, is this colonization the initial trigger for the localized inflammatory response or just aggravates the condition? With the available evidence it is hard to answer these questions. It has been proposed that atheromas might act as mechanical sieves, collecting bacteria from the cirulation (10). This would have deleterious consequences, as bacterial accumulation in the AP would lead to an increased inflammatory response. It could also link the fact that endotoxemia increases the risk of CVD (11), for which periodontal pathogens seem to play an important role (12). Supporting the role of infection as secondary to atherosclerotic inflammation, fungal DNA has been observed in AP (13), with some species correlated with that found on human microbial communities. It is of worth noting that in this study, fungal richness was not associated with classical CVD risk factors. Because not all normal residing oral bacteria are found in AP samples, AP invasion might be related to the virulence properties of some species (9). This seems to be the case, as in the study reviewed here, there was a common abundance of Streptococcus and Veillonella in AP samples. Streptococcus is able to adhere to the endothelium, while Veillonella is able to change its adherence capacity in the presence of some factors from Streptococcus (14). In fact, there is a tight relationship between Streptococcus and Veillonella in the oral cavity as some strains co-aggregate, partially because Veillonella seems to be metabolically dependent on Streptococcus (15). This relationship is so important that Veillonella is unable to establish an infection without Streptococcus.

In conclusion, microbial accumulation in AP might contribute to the progression of atherosclerosis. Although the mechanism by which these microorganisms colonize this site is not defined, it is clear that several microbes found in other body sites are also found in AP, which suggests that the normal human microbial communities are an important source of pathogens contributing to atherosclerosis progression. Translocation from these sites, in turn, is controlled by the host inflammatory status. This seems to be relevant to translocation from the oral cavity: transient bacteremia is experienced by everyone because of mechanical disruption of microbial biofilms (for instance, when eating), but it is controlled quickly. However, there are always some persisters which resist by-standing immune mechanisms. Obviously, a higher microbial load facilitates dissemination into the bloodstream and could possible influence the degree of transient bacteremia. A higher bacterial load coupled with a compromised subgingival epithelium barrier increases the risk of bacteremia and secondary colonization. So, in order to reduce the risk of AP colonization by oral pathogens, it is wise to target these two factors. For reducing oral bacterial load (overgrowth), reducing sucrose intake might be of benefit, as bacterial glucosyltransferase (GTF) plays a crucial role in plaque formation (16) and S.mutans is only pathogenic in the presence of sucrose (17). Dietary sucrose has been shown to increase total viable microbial density and S.mutans population in human dental plaque (18). Sucrose alone seem to be more cariogenic than sucrose plus fructose (19, 20). Additionally, sucrose alters the ionic concentration in the biofilms' matrix, altering the normal de- and re-mineralization process of enamel and dentin  (21). The role of starches in dental plaque formation is controversial (22), although some authors are in agreement with the Cleave & Yudkin hypothesis, which states that an excess of fermentable carbohydrate intake (in the absence of dental interventions) promotes dental diseases and then systemic diseases (23). Nevertheless, starchy foods commonly ate might promote dental plaque formation and disease. Pollard (24) showed that cornflakes, branflakes and wholemeal bread produced the minimum dental plaque pH peak, while all foods tested promoted enamel demineralization*. This might be related to the fact that, although starches can reduce plaque pH and induce demineralization, sucrose accelerates this effects (25). This is probably mediated by the interaction between bacterial GTF and salivary amylase (26). In contrast to what some might expect, whole fruit and fruit juices induce enamel demineralization by the same magnitude (27). This has been also found in some observational studies, where high fruit consumption is associated with increased caries risk (28). On the contrary, cheese and nuts have shown a negative association (29). Finally, inflammation increases the risk of oral bacterial growth and translocation, which might induce and/or aggravate systemic diseases (30). Periodontal disease has been positively associated with obesity (31), metabolic syndrome (32), type 2 diabetes (33), Alzheimer's disease (34), among other. Thus, controlling inflammation is key to avoid secondary diseases caused by pathogenic oral bacteria. 

* "Test foods were oranges, apples, bananas, Cornflakes, Branflakes, Weetabix, Alpen (no added sugar), white bread, wholemeal bread, rice, and spaghetti, with positive and negative controls of sucrose and sorbitol."

Koren O, Spor A, Felin J, Fåk F, Stombaugh J, Tremaroli V, Behre CJ, Knight R, Fagerberg B, Ley RE, & Bäckhed F (2011). Human oral, gut, and plaque microbiota in patients with atherosclerosis. Proceedings of the National Academy of Sciences of the United States of America, 108 Suppl 1, 4592-8 PMID: 20937873