Got Milk? Part 4: Into the Rabbit Hole!

A genetic trait, known as lactase persistence (LP), allows carriers of the gene to drink milk after they are weaned. The 'dairy councils' of the world, with the backing of the medical profession, want everyone drinking MOAR MILK. Our very own Gabriella Kadar, DDS, made the connection that possibly this lactase persistence gene was the root cause, or co-factor of a modern disease, multiple sclerosis.

Gabriella recently discussed this phenomenon with Gemma. Gemma, per usual, dug up many good papers and connected some dots that got Gabriella really scared! Possibly there's even more to the story than MS...

Yes!

From Gemma:

A couple of weeks ago Gabriella mentioned to me that she suspects lactase persistence gene / status has something to do with susceptibility to multiple sclerosis, a hypothesis she researched a few years ago, out of pure curiosity. Gabriella said she had noticed that all people with multiple sclerosis she knew were drinking milk.  

An original hypothesis on a disease with unknown cause? A purely human disease, with no exact animal equivalent? A modern day disease, first described in 1868? An autoimmune disease, with  very long latency up to 20 years? A disease, when a body is attacking its own tissues? A disorder of altered T-cell homeostasis? No single pathogen responsible for MS was ever found, no single causal gene was ever identified, HLA genes and vitamin D status cannot explain all, hygiene hypothesis is not enough, and the causes seem to be epigenetic, humans are the only mammals that keep LP after weaning... and MS might start in the gut (2014).

More reading:
The story of multiple sclerosis 
Multiple Sclerosis Review (2012)
Multiple Sclerosis: A Disorder of Altered T-Cell Homeostasis (2011)
Nutrition Facts in Multiple Sclerosis (2015)

Should be fun to research, I thought. In the worst, our findings would simply join the very long line of hypotheses, all exposing a gap in understanding of multiple sclerosis causality. The scientists managed to come up with many theories so far, some quite esoteric indeed. Is it exposure to domestic cats? Is it a lack of exposure to domestic cats? Is it house dogs? Is it volcanogenic microelements? No, we don't think so. So, we set out to explore the cause of multiple sclerosis.

Here is what we found so far:

LCT gene and Chromosome 2 

First we had a look at Gabriella's suspicion that lactase gene (LCT) is just a red herring, that there is actually another faulty gene in its surrounding, in an area called 2q21, on a longer arm of the chromosome 2. This chromosome is very suspicious, since it is considered to be a fused chromosome, and the remnants of the ancient centromere are exactly in our region of interest, close to 2q21 where LCT is located. Human Chromosome 2 "is widely accepted to be a result of an end-to-end fusion of two ancestral chromosomes, as evidenced by the presence of a vestigial centromere. Normally a chromosome has just one centromere, but in chromosome 2 there are remnants of a second centromere. Moreover, in chromosome 2 there are additional telomere sequences in the middle."

More reading:
Origin of human chromosome 2: an ancestral telomere-telomere fusion. (1991)

Such a region might be a bit unstable and some more frequent mutations could be there. We found crazy many interesting genes at 2q21 or nearby, some even suspected to be involved in the pathogenesis of multiple sclerosis this way or another, but no single gene seemed to be The One. That does not mean that a deleterious mutation hitchhiking together with a positively selected allele for LP is not there, waiting to be discovered. 

GWAS studies looking for a causal gene(s), implicated in MS

Was LP gene ever identified as suspicious in MS? Genome-wide association studies (GWAS) are often used to study the DNA of groups of people. Comparisons are made between people with a particular disease and those without to see what genetic clues can be learned. Funny fact is that LCT gene is a strong confounder in GWAS studies for multiple sclerosis (MS), and as such removed from considerations.  This points to something that Gabriella picked up on.

More reading:

Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls (2007), see Table 1

Confounding Underlies the Apparent Month of Birth Effect in Multiple Sclerosis (2013)

Some scientists are starting to "see the light" at the end of the tunnel, as far as LP and lactose is concerned, but we are faster!

More reading:
Latitude, sunshine, and human lactase phenotype distributions may contribute to geographic patterns of modern disease: the inflammatory bowel disease model (2014)

Adult lactose digestion status and effects on disease. (2015, in press)

 The implications of lactase persistence

Another thought we had reflected an idea that MS might be connected to the lactase gene being used and turned ON, even after weaning. So our attention turned to milk drinking and LCT gene itself, and we started to explore what it is actually good for, and why humans developed lactase persistence (LP) beyond weaning age. Some facts summarized in Part 1 and Part 2 of this series.

More reading:
T-13910 DNA variant associated with lactase persistence interacts with Oct-1 and stimulates lactase promoter activity in vitro. (2005)
Analysis of a SNP linked to lactase persistence: An exercise for teaching molecular biology techniques to undergraduates (2011)

Consult wiki or other literature, if you need to learn more about lactose (the milk sugar, a disaccharide sugar derived from galactose and glucose) or lactase, "also known as lactase-phlorizin hydrolase, or LPH, a part of the β-galactosidase family of enzymes, is a glycoside hydrolase involved in the hydrolysis of the disaccharide lactose into constituent galactose and glucose monomers."

But what exactly made LP possible? Was natural selection in play? And who is the puppet master controlling evolution and the genome change? Seems it is our beloved microbes...

The hologenome theory of evolution

Role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution. (2008)

We present here the hologenome theory of evolution, which considers the holobiont (the animal or plant with all of its associated microorganisms) as a unit of selection in evolution. The hologenome is defined as the sum of the genetic information of the host and its microbiota. The theory is based on four generalizations: (1) All animals and plants establish symbiotic relationships with microorganisms. (2) Symbiotic microorganisms are transmitted between generations. (3) The association between host and symbionts affects the fitness of the holobiont within its environment. (4) Variation in the hologenome can be brought about by changes in either the host or the microbiota genomes; under environmental stress, the symbiotic microbial community can change rapidly. These points taken together suggest that the genetic wealth of diverse microbial symbionts can play an important role both in adaptation and in evolution of higher organisms. During periods of rapid changes in the environment, the diverse microbial symbiont community can aid the holobiont in surviving, multiplying and buying the time necessary for the host genome to evolve. The distinguishing feature of the hologenome theory is that it considers all of the diverse microbiota associated with the animal or the plant as part of the evolving holobiont. Thus, the hologenome theory fits within the framework of the 'superorganism' proposed by Wilson and Sober.

 

Natural selection and the immune system

Why do organisms adopt new genetic changes? Natural selection favors those who have more viable offspring, showing reproductive fitness, in other words. Fitness also stands for an ability to resist pathogens, so the immune system continuously evolves too, as yet another new pathogen shows up and tries to out-trick us. Gene network involved in the immune system function can be seen as a result of eons of battles, losses and victories against age-old pathogens that we are not perhaps facing in our modern times any more. But the immune system is still ready to fight them, in case they show up, or more importantly, when it perceives they might have shown up. Our immune system watches for signals that something is wrong, it watches for signals of stress and danger.

More reading:
A holobiont birth narrative: the epigenetic transmission of the human microbiome (2014)
The gut microbiome shapes intestinal immune responses during health and disease (2009)

Human nutrition, the gut microbiome, and immune system: envisioning the future (2011)

What does milk have to do with the immune system? Well, a lot! We know that milk is packed with compounds not only feeding and nourishing a neonate, but protecting and defending the infant whose immune system is not yet fully developed. Some researcher even suggest that the nutritional role of milk evolved secondarily to its immune function, see: Evolution of the mammary gland from the innate immune system? (2006).

We said that immune system is watching for signs of stress. And as we know, our commensal microbiota is watching too, from the point of view of the superorganism, and evaluating: "is that what we together agreed upon, when adopting a new genetic change, still respected?"

In other words: does  adoption of LP into the human genome mean the microbiota, the puppet master, says: "OK, my dear human, you can continue to eat some dairy even after weaning, but make sure that all that the toxins are neutralized and do not affect us! Make sure that toxic lactose is hydrolyzed, so that we both can enjoy some more easily digestible sugars and benefit from more energy. Have some lactose metabolizing microbes around, just in case something bad happens to your own lactase enzyme. But we are telling you, do not dare to lose both, and do not bomb us with toxins. It would make us quite hungry. And angry too."

Is milk a suspect in multiple sclerosis?

Let's go back to the autoimmune disease we are researching: multiple sclerosis. Have other researchers ever explored milk and MS connection? This paper seems promising, but no satisfactory explanation is offered...

Milk consumption and multiple sclerosis--an etiological hypothesis. (1986)

Epidemiological studies of Multiple Sclerosis (MS) in the United States have shown an association with urban living and higher socio-economic groups and a higher incidence and earlier age at onset of symptoms in women. This study is based on the proposition that these trends may be a consequence of differences in exposure to an etiological factor around 15 years of age. As a result of variations in related United States and New Zealand data and other pertinent observations the possibility of a link between high childhood milk intake followed by a large or sudden reduction during the adolescent growth spurt, and the subsequent incidence of MS in young adults is proposed. The possible involvement of calcium and lead metabolism is also discussed. It may be that the elusive environmental variable associated with the incidence of MS is partly a behavioural one related to western social attitudes.

Here is another paper, getting a bit closer, but still identifying a wrong target, in our opinion:

Correlation between milk and dairy product consumption and multiple sclerosis prevalence: a worldwide study. (1992)

Multiple sclerosis (MS) epidemiology suggests that different factors are involved in the clinical expression of the disease. Alimentary cofactors have already been considered, but mainly theoretically. We have studied the relationship between MS prevalence and dairy product consumption in 27 countries and 29 populations all over the world, with Spearman's correlation test. A good correlation between liquid cow milk and MS prevalence (rho = 0.836) was found; this correlation was highly significant (p < 0.001). A low but still significant correlation was obtained with cream or butter consumption (rho = 0.619 and rho = 0.504, respectively). No correlation was found for cheese. These results suggest that liquid cow milk could contain factor(s) - no longer present in the processed milk - influencing the clinical appearance of MS. The possible role of some dairy by-products is discussed in the light of a multifactorial etiology of MS.

This paper from 1992 identifies the highly significant trigger present in liquid milk, but suggest that the milk component responsible for triggering MS is, surprisingly, butyrate. This conclusion made us laugh, really. No, it cannot be our beloved butyrate. So, which other factor present in liquid milk, and not anymore present in processed milk, could it be?

Innocent lactose?

We are mainly interested the immunomodulatory function of milk, so let's have a closer look at it. What is one of the strongest inducers of innate immunity in mammalian milk? This new research suggests that it is our milk sugar of interest, lactose. We have mentioned that lactose is selectively toxic to gut flora. On the other side, baby gut flora, namely Bifidos, enjoy lactose quite a lot, actually so much that they metabolize lactose preferably over glucose. But what about other microbes? Oh, oh...This paper revealing hitherto unknown function of lactose in innate immunity says:

Lactose in Human Breast Milk an Inducer of Innate Immunity with Implications for a Role in Intestinal Homeostasis (2013)

Postpartum, infants have not yet established a fully functional adaptive immune system and are at risk of acquiring infections. Hence, newborns are dependent on the innate immune system with its antimicrobial peptides (AMPs) and proteins expressed at epithelial surfaces. Several factors in breast milk are known to confer immune protection, but which the decisive factors are and through which manner they work is unknown. Here, we isolated an AMP-inducing factor from human milk and identified it by electrospray mass spectrometry and NMR to be lactose. It induces the gene (CAMP) that encodes the only human cathelicidin LL-37 in colonic epithelial cells in a dose- and time-dependent manner. The induction was suppressed by two different p38 antagonists, indicating an effect via the p38-dependent pathway. Lactose also induced CAMP in the colonic epithelial cell line T84 and in THP-1 monocytes and macrophages. It further exhibited a synergistic effect with butyrate and phenylbutyrate on CAMP induction. Together, these results suggest an additional function of lactose in innate immunity by upregulating gastrointestinal AMPs that may lead to protection of the neonatal gut against pathogens and regulation of the microbiota of the infant.

 Some other interesting facts, worth noting:

It is well known that lactose is first hydrolyzed into its glucose and galactose moieties by lactase, located in the mucosa of the small intestine [32]. Our results demonstrate no detectable levels of lactose in fecal samples of both one and four week old infants (Table 1). This suggest an efficient hydrolysis of lactose in the small bowel, which is in agreement with previous reports [33]. However, we cannot rule out the possibility that a part of the lactose is internalized by the colonic epithelial cells.

In addition to lactose, the levels of galactose were determined in the same feces samples and were found to be 1–7 g/l (Table 1), revealing an incomplete absorption of this monosaccharide in the gut. Interestingly, we observed that an equimolar concentration of a combination of glucose and galactose induced CAMP gene expression to the same extent as lactose. Furthermore, our results indicate that also maltose induced CAMP gene transcript to the same extent as lactose and that trehalose can even surpass lactose in CAMP gene-inducing capacity on an equimolar basis.

Now, what exactly is cathelidicin (also called CAMP or LL-37)? A very ancient antimicrobial peptide, acting as endogenous antibiotic, produced by the innate immune system. So ancient that the pathogens learned to live with it, and sometimes can even hijack its action to promote their own survival.

"So what?" you say. LL-37 can be upregulated by many other factors. What else can induce expression of  LL-37? Well, for instance, bacterial products, butyrate, or vitamin D, among others. Is upregulated LL-37 good or bad? It depends. It is good if you need to fight an infection and kill the pathogenic microbes. But what if they are not there?

More reading:
Little peptide, big effects: the role of LL-37 in inflammation and autoimmune disease (2013)
The antimicrobial peptide LL37 is a T-cell autoantigen in psoriasis. (2014) 

Immune system in overdrive?

Can it get any worse? What else can the innocent milk sugar lactose do, when it escapes digestion either by low/no activity of endogenous lactase, or by missing gut flora? Note that this is seen as a defect, it should not really happen too often. If there is too much lactose around, our immune system might think that something is wrong, and start an attack! Or, a smart commensal microbe afraid of lactose or of the imaginary pathogen might do the same...

See this in vitro research:

Lactose inhibits regulatory T-cell-mediated suppression of effector T-cell interferon-γ and IL-17 production (2014)

Our interest in lactose as an immunomodulatory molecule results from studies showing that lactose binds to galectin-9, which has been shown to have various regulatory functions in the immune system including regulation of T-cell responses. Impaired regulation of T helper (Th)1 and Th17 type immune responses and dysfunction of regulatory T cells (T_reg) have been implicated in many human immune-mediated diseases. In the present study, we investigated the effects of lactose on immune regulation using co-cultures of human peripheral blood mononuclear cell (PBMC)-derived T_reg and effector T cells (T_eff) obtained from twenty healthy adults. T_reg, i.e. CD4⁺CD25⁺CD127⁻, were isolated from PBMC by immunomagnetic separation. The fraction of CD4⁺CD127⁻ cells that was depleted of CD25⁺ cells was used as T_eff. T_reg and T_eff at a ratio 1:5 were activated and the effects of lactose on the secretion of interferon-γ (IFN-γ) and IL-17 were analysed using ELISA for protein and quantitative RT-PCR for mRNA. T_reg down-regulated the secretion of both IFN-γ (8·8–3·9 ng/ml, n 20, P= 0·003) and IL-17 (0·83–0·64 ng/ml, n 15, P= 0·04) in co-cultures, while in the presence of lactose the levels of secreted IFN-γ and IL-17 remained high and no down-regulation was observed (16·4 v. 3·99 ng/ml, n 20, P< 0·0001, and 0·74 v. 0·64 ng/ml, n 15, P= 0·005, respectively). We showed that lactose inhibits human T_reg-mediated suppression of Th1 and Th17 immune responses in vitro.

Let's sum it up: 

This basically describes the altered T-cell homeostasis, mentioned as one of the MS attributes. Per previous paper, lactose can induce expression of endogenous antibiotic (LL-37) and at the same time induce pro-inflammatory response and block the anti-inflammatory Tregs that are important in the maintenance of immune homeostasis. Autoimmunity landscape par excellence.

The authors conclude with these points:

• ...it is plausible that in individuals susceptible to chronic inflammatory diseases, dietary lactose could induce harmful inflammatory responses by disrupting Treg-mediated regulation...
• The incidence of autoimmune diseases, chronic inflammatory disorders and allergy has increased during the last few decades, especially in Western societies, and cannot be explained by changes in genetic predisposition.
• Uncontrolled Th1 and Th17 immune responses and the inability of Treg to down-regulate immune responses have been implicated in the pathogenesis of many human immune-mediated diseases.
• ...the role of dietary lactose in the exacerbation of allergic inflammation in individuals with food allergy should be considered.
• In populations with a high proportion of lactose-tolerant individuals, due to the high frequency of lactase gene mutation contributing to the persistent intestinal lactase production during adulthood, lactose intake from the diet is relatively eminent.
• Interestingly, the incidence of some immune-mediated diseases is high among these populations.
• It is also possible that the intestinal problems associated with genetic lactose intolerance may not always be caused by lactose-related osmotic changes, but could be of immunological origin.
• Taken together, lactose has strong immune-modulating properties, which we have demonstrated in the present study in vitro in human subjects...
• Physiological relevance of our preliminary results and effects of dietary lactose on the human gut immune system and health need to be studied further.

Can inappropriate lactose signaling during dysbiosis, or in the absence of pathogens, cause an inappropriate immune response? Back in the "good old days" when we were surrounded by pathogens and forced to eat dirt with our meals, perhaps an immune system in overdrive was advantageous. Now, in our semi-sterile lives, drinking milk is still a signal that keeps our immune system in hyperdrive.

Babies are full of Bifidobacteria. Bifidobacteria is associated with a healthy baby. Before we started drinking the milk of other species, we were forced to develop an adult microbiota. Continually feeding our gut bacteria "baby food" may have profound implications when attempting to shift the gut flora from that of a baby's to that of an adult.

More reading:
The contribution of natural selection to present-day susceptibility to chronic inflammatory and autoimmune disease. (2014)
Regulation of osteoclastogenesis through Tim-3: possible involvement of the Tim-3/galectin-9 system in the modulation of inflammatory bone destruction. (2014) 

Evolution has no eyes to the future!*

*(Williams GC. Adaptation and Natural Selection. Princeton, New Jersey: Princeton University Press; 1966.)

Selective pressure does not look kindly upon humans acting against their own genes. Over the past several million years, we've perfected our immune systems and physiology. Now these mismatches in milk drinking, on a very new genotype, are creating "weak points" in our lives. We drink lactose, signalling a need for an immune boost, but with few pathogens present.  Perhaps when we had a high pathogen load, bellies filled with helminths and H. pylori, being LP was truly advantageous and milk allowed us to thrive.

But, no traditional means of milk processing gave anyone much lactose. Traditional methods all were lactose reducing. Cheese, fermented milk, and even the occasional drink of raw, not pasteurized or cooled and stored milk were the methods of dairy consumption that allowed humans to become LP. Nowadays, we drink all manner of processed, high-lactose milk products whenever we like. As Gabriella noticed, the LP genotype/status plays a BIG ROLE in susceptibility to modern inflammatory/autoimmune diseases, not only to multiple sclerosis. Researchers noticed it, too, but only at the genetic level, never connecting the problems with the lactose itself.

What Gabriella has hypothesized makes perfect sense. A gene or haplotype with a role in reproductive fitness connected to an ability to digest milk past the age of weaning. Milk is simply an external input to our innate immune system. Babies get their first microbes from their mothers, the mother also fuels their immune system with milk.

When Gabriella noticed the connection between the veritable milk orgy we've been engaged in since the advent of pasteurization and an explosion of MS, she was seeing a nasty trick of evolution at play. What was protective for humans when we were living a hard-scrabble life of recently liberated hunter-gatherers is deadly for modern humans. When the immune system ramps up for no good reason, it attacks anything it perceives as a threat. In auto-immune disease, that threat is us.

Some thoughts...

Dr. Kadar brilliantly noted a connection that few researches have seen. That lactose and the drinking of milk by entire populations of people without regard to the genetics of LP or LNP is most likely causing the MS we see in the world today, and possibly other autoimmune diseases too. What is T1D, anyway? We're calling for research in this area! The data has mostly been collected, it just has not been screened for this scenario.

We further hypothesize that the drinking of fluid, pasteurized milk in quantities only seen since the 1800's has overridden the genetic ability of our immune system to use lactose as a signalling molecule, and we suggest that the lactose is the problem. 

Does Multiple Sclerosis start in the gut, is microbiota involved?

We think so. But not in the way researchers tend to look at it, searching for a unknown evil pathogen with a causal role. We have stated that our immune system watches for signals that something is wrong. But so does our guardian microbiota that has co-evolved in tight relationship with us and our immune system. Therefore, we hypothesize that an ordinary, commensal microbe may have a causal role in autoimmunity.

Molecular Mimicry Revisited: Gut Bacteria and Multiple Sclerosis (2006)

Molecular mimicry is a possible explanation for autoimmune side effects of microorganism infections. Protein sequences from a particular microorganism are compared to known autoimmune immunogens. For diseases such as multiple sclerosis (MS), where the infectious agent is unknown, guesses to its identity are made. Mimics are assumed to be rare. This study takes a radically different approach. Reported sequences from all known human bacterial and viral agents were searched for autoimmune immunogen mimics. Three encephalitogenic peptides, whose autoimmune requirements have been studied extensively, were selected for comparison. Mimics were seen in a wide variety of organisms. For each immunogen, the mimics were found predominantly in nonpathogenic gut bacteria. Since the three immunogens used in this study are related to MS, it is suggested that a microorganism responsible for autoimmune activity in MS could be a normally occurring gut bacterium. This would explain many of the peculiar MS epidemiological data and why no infective agent has been identified for MS and supports recently found MS gut metabolism abnormalities.

But a microbe and the immunogen is not enough...

For molecular mimicry to work the immunogen must be “visible” to the immune system. It has therefore been assumed that it must be located on the surface of a protein that is either excreted by the microorganism or is on its surface. This is not necessarily true. The microorganism itself can initiate an immune response. Cells are hydrolyzed, and their contents are released. A great amount of digestive enzyme activity is present. All this can “expose” potential immunogens (23). It certainly happens within active EAE lesions. These “new” immunogens can be processed by the already-present immune cells.

And what else is missing?

Why is the incidence of MS so low? Although both the immunogen and adjuvant molecules are present, they must be processed in such fashion that the correct units are made in high enough concentration to form the complex. This will depend entirely upon the nature of the gut and its hydrolytic enzymes. The gut is a dynamically evolving biosystem whose bacterial content is changing and therefore the concentrations and types of hydrolytic enzymes continually vary.

Conclusions and open questions

We propose that an innocent commensal microbe, perturbed by lactose signalling during dysbiosis, starts a causal cascade to autoimmune disease.  Which one? Does it matter? Does bacterial beta-galactosidase play a role too? What is the initial trigger setting a stage for autoimmune attack on myelin? We do not know, we only have some wild ideas... But we hope for more research in this area soon!

More reading:
Intestinal Barrier Dysfunction Develops at the Onset of Experimental Autoimmune Encephalomyelitis, and Can Be Induced by Adoptive Transfer of Auto-Reactive T Cells (2014)
Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis (2010)
Commensal microbiota and myelin autoantigen cooperate to trigger autoimmune demyelination.(2011)