Monkey’s Uncle? Your HLA might be Neanderthal

Humans’ genomes are extremely similar to those of other primates because the species diverged relatively recently, approximately 6 million years ago in the case of our nearest cousins, Chimpanzees. Modern humans and Denisovans separated 250,000 years ago (10,000 generations). With the recent sequencing of extinct, ancient hominids, such as Neanderthals and their Denisovan relatives, it was realized that up to 6% of the genomes of humans now in Europe and Asia derive from these older lineages.

HLA genes are by far the most polymorphic within the human genome, with thousands of variants (alleles). Here, investigators first identified one particular HLA allele, HLA-B*73:01, as being more similar to homologous Chimpanzee alleles than other human HLA-B alleles. This allele diverged from other HLA-B alleles 16 million years ago, before the separation of humans and Chimps, and was lost from the majority of modern humans. Its reappearance in the human genome was most likely, they reckoned, a result of “introgression”, introduction from ancient humans such as Neanderthal. An alternative model, which computer simulations indicate is 100 times less probable, is that this allele came out of Africa late

They also simply “typed” (sequenced and matched) the most important HLA loci, HLA-A, -B and –C from 1 Denisovan and 2 Neanderthal subjects. Surprisingly, most of these archaic HLA alleles were identical to common HLA types of modern humans. HLA-A2, the most widespread allele at the HLA-A locus, was shared with and might have been acquired from Denisovans. Putative archaic HLA-A alleles are now more common in China and Europe than in Africa (Figure, from fig. 4d). The authors conclude that although a small minority of our genomes overall derived from archaic humans, about half of our HLA was acquired through interbreeding between modern humans migrating out of Africa and locally established archaic humans. These archaic alleles conferred fitness in the new environment, e.g., pathogen and allergen resistance, and so outcompeted and displaced previous human HLA alleles.


The shaping of modern human immune systems by multiregional admixture with archaic humans. Abi-Rached et al. Science. 2011 Oct 7;334(6052):89-94.

Gut Feeling – intestinal microbes influence immune system tolerance of central nervous system

Multiple Sclerosis (MS: Wikipedia, PubMedHealth) is an autoimmune disease wherein lymphocytes attack the central nervous system (CNS), including the brain and spinal cord, leading to relapsing, progressive loss of neurons. Lesions containing B and T lymphocytes develop in the CNS. The cause of MS is unknown.

A mouse model of MS, called experimental autoimmune encephalomyelitis (EAE), can be induced when mice of certain strains are immunized with spinal cord proteins, or it can occur spontaneously in genetically engineered strains in which many CD4+ “helper” T cells express a transgenic T cell receptor specific for myelin oligodendrocyte glycoprotein (MOG), a protein abundant on the surface of key non-neuronal cells of the CNS.

These authors observed that depending on the animal housing facility, between 35-90% of MOG-specific-TCR-transgenic mice spontaneously develop EAE at between 3-8 months of age. The wide range in the disease incidence reminded the authors of a 1993 report by Goverman that mice with T cells expressing transgenic antigen receptors specific for another nerve protein, MBP, developed EAE ‘spontaneously’ in non-sterile housing but not in sterile housing.

They compared EAE incidence in mice that possess normal gut microbes but harbor no known pathogens, termed Specific Pathogen Free (SPF), and mice that possess no microbes at all, termed “germ free” (GF), and found that GF mice were protected (Fig 1a, shown, left panel).

Gut microbes are known to contribute to lymphocyte maturation, stimulated by , e.g., segmented filamentous bacteria) or polysaccharides of Bacteroides fragilis. However, the authors argue this does not explain protection because GF mice colonized with “conventional commensal” microbes developed EAE “promptly”, starting about a month later (Fig 1b, shown, right panel). They add that colonization with segmented filamentous bacteria – shown to trigger autoimmunity in another model – conferred EAE susceptibility only inefficiently. They also argue that GF mice immunized with MOG in complete adjuvant develop EAE (though again with a delay of about a month) and produce specific antibodies (though measured crudely, not titered), demonstrating that their lymphocytes are mature.

Instead, the authors argue that some lymphocyte activities are reduced in GF mice, particularly T cell production of the pro-inflammatory interleukin-17 and spontaneous B cell production of MOG-specific antibodies (which is also “promptly” albeit only partially corrected by colonization, Fig 3a). Moreover, MOG-specific B cells – but not polyclonal normal B cells – transferred into MOG-specific-TCR-transgenic mice – but not MOG-deficient mice – home to germinal centers where they mature and make antibodies that are IgG2a class-switched, and therefore implicitly effective in cooperating with specific T cells to induce EAE. They conclude that commensal gut microbes activate autoreactive T cells that recruit autoreactive B cells, which together mediate disease.

Berer K, Mues M, Koutrolos M, Rasbi ZA, Boziki M, Johner C, Wekerle H, Krishnamoorthy G. Commensal microbiota and myelin autoantigen cooperate to trigger autoimmune demyelination. Nature. 2011 Oct 26;479(7374):538-41. doi: 10.1038/nature10554. PubMed PMID: 22031325

Would you like some E. coli with that?

An epidemic of bloody stools and failing kidneys, some with hemolytic uremic syndrome (HUS) appeared in Germany in May 2011 and subsequently 15 other countries. By late July when the epidemic had subsided, a total of 3,816 cases - including 54 deaths - were reported in Germany, 845 of which included HUS. Rasko and colleagues cultured E. coli bacteria isolated from a 64 year old woman from Hamburg, Germany, who did not develop HUS. They characterized this bacterium, designated C227-11, as enteroaggregative, which means a gut pathogen that aggregates and forms “biofilms” that are resistant to treatment.

They sequenced the bacterium’s genome and found it was a unique strain of the O104:H4 serotype of E. coli bacteria, distinguished by possession of a prophage (http://en.wikipedia.org/wiki/Prophage ) producing the Shiga toxin. Shiga toxin binds to cells, inhibits protein synthesis, and kills by inducing apoptosis [review]. The O104 serotype is rare; the most frequent cause of HUS worldwide is the shiga-toxin–producing E. coli O157 (Tarr 2005).
Although they isolate only one strain themselves, they analyzed also 3 additional sequences from the current outbreak that had been made public (that’s data mine-ing!) together with 7 other O104:H4 serotype isolates, all from Africa, and 4 other reference strains. The authors conclude that the outbreak was caused by a difficult (enteroaggregative) strain made more virulent by its acquisition of the Shiga toxin gene in addition to antibiotic-resistance and “additional virulence and antibiotic-resistance factors”. Rohde and colleagues reached the same conclusion using "rapid, bench-top DNA sequencing technology, open-source data release, and prompt crowd-sourced analyses".

Where did the E. coli O104:H4 come from? A subsequent publication reported the results of trace-back and –forward investigations by Buchholz and colleagues who analyzed 26 HUS patients and 81 healthy controls. They concluded that despite only about a quarter of the patients recalling in exploratory interviews having eaten bean sprouts during the 14 days before the onset of illness, 100% of these illnesses were attributable to the consumption of sprouts – and not other raw foods such as tomatoes or cucumbers or lettuce – at a particular restaurant, and for other patients, sprouts obtained from a single, common supplier (figure).

N Engl J Med. 2011 Aug 25;365(8):709-17. Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany. Rasko DA, Webster DR, Sahl JW, Bashir A, Boisen N, Scheutz F, Paxinos EE, Sebra R, Chin CS, Iliopoulos D, Klammer A, Peluso P, Lee L, Kislyuk AO, Bullard J, Kasarskis A, Wang S, Eid J, Rank D, Redman JC, Steyert SR, Frimodt-Møller J, Struve C, Petersen AM, Krogfelt KA, Nataro JP, Schadt EE, Waldor MK.

Helpful mutants usually won’t cooperate with each other

How genes interact – the phenomenon termed “epistasis” – is complex, yet must be understood to accurately interpret the contribution of individual genes to the development and behavior of the organism. Two groups -- Chou et al. and Khan et al. -- recently reported in Science the results of their remarkably similar experiments; they isolated individual, beneficial mutations in bacteria then tested how the mutations interact when possessed by the same bacterium.

Previous studies (cited by Chou) suggested that two deleterious mutations in the same pathway are generally less-than-additive but were greater-than-additive when they were in parallel pathways, which seems intuitive. Interactions between mutations in single genes were shown to depend on the background (other genes). However, epistasis among beneficial mutations in different genes was “unexplored”, and the focus of these new experiments. Evolution in laboratory conditions is initially rapid but quickly slows, which fits a model of mutually antagonistic beneficial mutations. Khan states the deceleration is due to either (1) negative epistasis or (2) because the most beneficial would “tend to be incorporated earlier owing to their faster spread and greater success in the face of competing beneficial mutations” (which sounds suspiciously convenient – the best arrive early – but they give a reference: Gerrish 1998 so you can look it up).

Khan grew E. coli with a glucose supplement for 20,000 generations, when they sequenced a clone and identified 45 mutations. They say other beneficial mutations arose but were lost due to “interference” with more-beneficial mutations (24, 26) (which seems to answer their question), or because they were “less able to evolve than the eventual winners” (33). They took the first 5 “that fixed … and whose spread coincided with the period of fastest adaptation” (arbitrary? How many generations was that, 200? 2,000?), which were, in order of appearance: rbs operon, topA, spoT, glmUS promoter, and pykF. Together, these 5 mutations increased fitness ~30%, accounting for ~80% of the fitness increase over the full 20,000 generations.

Then they produced 32 populations of E. coli, one for each possible combination of the 5 genes (Fig. 1 from Khan, copied here, shows the ancestral genotype at the top and the 32 combinations of mutations, with increasing fitness downward). They found that although each combination improved fitness, improvement was less than expected from a “multiplicative null model” (in which the individual fitness effects are multiplied).

Similarly, Chou selected a bacterium to grow with methanol as its sole carbon source and then replaced a key metabolic pathway with a less efficient pathway from a different bacterium. The resulting bacterium grew only one-third as fast as the original. Eight separate populations improved the efficiency over 600 generations. In the fittest strain, 9 mutations were identified; 3 were clearly related to the pathway while 6 others were deemed “unlikely” to contribute to fitness. To determine the interactions among these 3 genes, they constructed 16 strains, one with each combinations of these 3 mutations plus the WT allele. They also observed that combination strains were much less fit than expected if they were acting independently (our old favorite, the multiplicative null model).

These findings should influence our expectations in genetic studies in humans; e.g., two disease-risk alleles may have little additive or even negative effects (ergo protective?), contradicting the simple expectations of most biologists. [Chou distinguished beneficial from detrimental effects.] I wonder if these experiments might be misleading because they first fixed the individual mutants, thereby eliminating mutants that interact positively during selection.

Khan AI, Dinh DM, Schneider D, Lenski RE, Cooper TF."Negative epistasis between beneficial mutations in an evolving bacterial population. " Science. 2011 Jun 3;332(6034):1193-6.

The same issue has a commentary.

Kidney disease linked to putative autoantigen + HLA

Idiopathic membranous nephropathy is a progressive disease involving the thickening of the basement membranes in glomeruli, which are key blood filtration units in the kidneys. Membranous nephropathy can be caused by exposure to toxins (gold, mercury, some medicines) or autoimmunity, such as lupus. Deposits of antibody-antigen (immune) complexes with complement components can be observed in the glomerulus.

These authors sought genetic associations in 556 biopsy-proven patients (British, French, & Dutch) by comparison of about 300,000 SNPs with matched healthy subjects. They found strong associations with a membrane protein previously implicated in autoimmunity, M-type phospholipase A2 receptor (PLA2R1, p~10E-28), and a histocompatibility gene (HLA-DQA1, p~10E-92). PLA2R1 is normally expressed in human glomeruli, exactly where immune complexes are found in membranous nephropathy patients. PLA2R1 was implicated only recently in autoantibody studies (Beck 2009) and is now known as the major autoantigen in idiopathic membranous nephropathy. The DQA1 association is not surprising, having been discovered by Vaughn et al using the relatively crude restriction fragment length polymorphism (RFLP) analysis and reported way back in 1989. The odds ratio for a single PLA2R1 risk allele is about 2 and for HLA-DQA1 about 6, modest but typical for such association studies. It is therefore astonishing that the risk to individuals possessing homozygous risk alleles at both loci is practically determinate – a 78.5-fold increased risk! – with 42 patients out of the 55 subjects possessing this combination.


How might this happen? The authors’ model is that perhaps the DQA1 molecule binds the PLA2R1 variant peptide and triggers T lymphocytes to help B cells make anti-PLA2R1 autoantibodies that bind to glomerular cells. They could have looked whether any PLA2R1 peptides has anchor residues that determine whether they can fit one of the 35 different allelic forms of DQA1. However, as Segelmark points out in the accompanying review, the strongest SNPs lie within the first introns for both PLA2R1 and DQA1 and (therefore) do not alter the amino acid sequence! The authors discount this, speculating that either the associated SNP is tightly linked to a variant that does change the protein. They could have sequenced the few subjects to identify any rare variant. Segelmark proposes as “more likely” that the SNP changes a regulatory sequence, such as a transcription factor binding site or a microRNA, that increases production of the proteins. A few more facts could help resolve these possibilities.
Risk HLA-DQA1 and PLA(2)R1 alleles in idiopathic membranous nephropathy. Stanescu HC, Arcos-Burgos M, Medlar A, Bockenhauer D, Kottgen A, Dragomirescu L, Voinescu C, Patel N, Pearce K, Hubank M, Stephens HA, Laundy V, Padmanabhan S, Zawadzka A, Hofstra JM, Coenen MJ, den Heijer M, Kiemeney LA, Bacq-Daian D, Stengel B, Powis SH, Brenchley P, Feehally J, Rees AJ, Debiec H, Wetzels JF, Ronco P, Mathieson PW, Kleta R. N Engl J Med. 2011 Feb 17;364(7):616-26.

HLA is the Major HIV Controller (MHC)

Upon infection by HIV, there follows a massive but transient increase in viruses in the blood followed by a period of years during which CD4+ T lymphocytes slowly decline. However, after the initial spike, some people have far fewer viruses in their blood and their CD4 counts do not decline. They remain apparently healthy, asymptomatic, for a very long time. These people are called “controllers” because their immune system controls virus replication without medication. The authors of this paper – with an impressive number of collaborators – looked for genetic variations in the controllers that could underlie the differences.

A genome wide association study (GWAS) performed with 1, 974 controllers (cases) and 2,648 progressors revealed a strong link with the HLA (the real human MHC, or major histocompatibility complex) on the short arm of chromosome 6 (figure shown). A weaker correlation with a chemokine receptor CCR5delta32 polymorphism previously identified with HIV resistance was also identified. HLA association is not surprising because practically everything immunological is strongly influenced by the HLA. What the investigators did next, however, was novel, imaginative, and highly illuminating.

From the SNP data, they were able to impute the controllers’ HLA type. Of the hundreds of HLA alleles, controllers tended to possess a remarkably narrow number of particular HLA-B and, to a lesser degree, HLA-A and -C alleles. They identified HLA-B*57:01, B*27:05, B*14/Cw*08:02, B*52, A*25 as protective alleles, and B*35 and Cw*07 as risk alleles. Moreover, they identified specific amino acids within the peptide-binding cleft as key variables in HIV control. These HLA amino acids define the ability of a pathogen to stimulate the cellular immune response.

These results provide a solid, well understood albeit complex mechanistic understanding to HIV progression and control. The next step could identify those HIV peptides that are bound preferentially by the protective and risk allele proteins.

The Major Genetic Determinants of HIV-1 Control Affect HLA Class I Peptide Presentation. The International HIV Controllers Study. Science. 2010 Nov 4.

Why Lupus can be Unresponsive to Glucocorticoid Therapy

Glucocoriticoids, including the natural hormone cortisol, are powerful immune suppressants. Synthetic glucocorticoids, such as dexamethasone and prednisone, are often taken by mouth to control autoimmune diseases. However, controlling the autoimmune disease systemic lupus erythematosus (SLE, lupus) often requires more aggressive treatments with high doses of more potent glucocorticoids, such as methylpredisolone, given intravenously.

Lupus is characterized by serum antibodies to nucleic acids (anti-nuclear antibodies, ANA), by a pattern of interferon-alpha-induced genes transcribed ("IFN signature”), and by an increase in plasmacytoid dendritic cells (PDC). PDC help produce antibodies and IFN. The IFN signature and PDC levels in patients are reduced to normal levels by high doses of intravenous glucocorticoids (see Figure 1c).

The authors noted that serum nucleic acids, which trigger the production of ANA, also stimulate PDC though specific receptors called toll-like receptors-7 and -9 (TLR-7 & -9) expressed on many cells of the immune system. Further, they hypothesized that TLR stimulation renders the PDC resistant to the suppressive effects of glucocorticoids.

Indeed, purified PDC treated with glucocorticoids (1-10 uM) do not survive overnight in a flask unless they are also treated with a nucleic acid (CpG) that stimulates TLRs (Figure 2a, first panel shown here). Nucleic acid-mediated protection is partially reversed by IRS, a synthetic oligonucleotide inhibitor of TLRs (IRS 954). Nucleic acid-containing immune complexes, isolated from the sera of lupus patients, also protected PDC by triggering TLRs.

Similar results were obtained with PDC from healthy people, suggesting that the PDC of lupus patients are not different but instead PDC are made glucocorticoid resistant by chronic stimulation of TLRs by nucleic acids. The authors conclude that “inhibitors of TLR7 and 9 signaling could prove to be effective corticosteroid-sparing drugs.”

Guiducci C, Gong M, Xu Z, Gill M, Chaussabel D, Meeker T, Chan JH, Wright T, Punaro M, Bolland S, Soumelis V, Banchereau J, Coffman RL, Pascual V, Barrat FJ. “TLR recognition of self nucleic acids hampers glucocorticoid activity in lupus.” Nature. 2010 Jun 17;465(7300):937-41.

Note: Similar results were reported by Lepelletier and colleagues at the Hopital Necker.

Old news: Targeting the Target of Rapamycin (TOR) to Stay Youngish

There are many strategies to remain healthy and reduce the effects of aging: healthy diet and exercise, yoga, drastically reducing calories, etc... all disappointingly slow and indirect. On the other hand, simply feeding mice the anti-rejection drug rapamycin has been shown to dramatically increase both the average and maximum life span, even when started late in life, probably by postponing death from cancer, by retarding mechanisms of aging, or both. Unfortunately, rapamycin also strongly suppresses the immune system, increasing the risk of a dangerous infection. How can we derive the benefits of rapamycin while avoiding the risks?
An endogenous inhibitor of the “Target Of Rapamycin” (TOR), and thus a candidate for the physiological rapamycin-like agent, are sestrins (Sesns), which are conserved proteins that are induced by cellular stress including by elevated adenosine monophosphate kinase (AMPK) or DNA damage.

Drosophila (fruit flies) have a single form of sestrin (dSesn). To define the activities of dSesn, these authors created flies with either gain- or loss-of-function dSesn mutations. Clever sleuthing with agonists and employing other mutant signaling molecules showed that TOR induces dSesn through reactive oxygen species (ROS, quenched by antioxidants such as vitamin E), jun-N-terminal kinase (JNK), and a transcription factor (FoxO). p53 was implicated but narrowly escaped the round-up of usual suspects. dSesn repaid TOR's attention by suppressing its stimulation of tissue growth. Thus dSesn is induced by TOR and suppress TOR activities, constituting a feedback inhibition system.
These authors go on to show that flies lacking dSesn suffer multiple age-related pathologies, including muscle degeneration and heart malfunction. These pathologies are prevented by activating AMPK with AICAR (5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside) or inhibiting TOR with rapamycin (Figure 5C shows that heart arrythmias suffered by dSesn-deficient flies are treated by feeding them AICAR, rapamycin, or vitamin E).

Forget caloric restriction, I'll take my ice cream with a statin mixin and Rapa sprinkles. Or a burger with heaping side order of sestrins.

JLee JH, Budanov AV, Park EJ, Birse R, Kim TE, Perkins GA, Ocorr K, Ellisman MH, Bodmer R, Bier E, Karin M. “Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies” Science 2010 Mar 5;327(5970):1210-1.

Cholesterol-retentive Leukocytes

Elevated white cells in the bloodstream (leukocytosis) was correlated with cardiovascular disease and atherosclerosis nearly a century ago but the cause remains unclear. Elevated high-density lipoprotein (HDL), in contrast, is correlated with protection from cardiovascular disease, in part because HDL carries cholesterol away from macrophages in atherosclerotic plaques. Here, Yvan-Charvet and colleagues report that leukocytosis develops in mice lacking the membrane proteins ABCA1 and ABCG1 that normally transport cholesterol out of macrophages to lipoproteins. These ABCA1/G1-deficient mice suffer a myeloproliferative disorder and display an expansion of particular blood-forming (hematopoietic) stem cells. Although lymphoid (B, T, NK cells) precursor cells were unchanged, myeloid precursor cells, which give rise to granulocytes, macrophages, etc, were doubled. Similar findings in MyD88-knockout mice ruled out innate inflammation as a cause of leukocytosis. Transplantation of bone marrow from ABCA1/G1 transporter-deficient mice into (apoA1-transgenic) mice with elevated HDL blocked or slowed the development of leukocytosis, myeloproliferation, the particular stem cell population, and atherosclerosis. The figure shows that elevated HDL in apoA1-tg mice protected against heart disease (Fig 4B, left side shows the entire hearts and, right side, tissue sections from hosts transplanted with ABCA1/G1-deficient bone marrow; upper panel: diseased heart from a normal recipient mouse showing leukocyte infiltration and, lower panel, healthy heart from a HDL-elevated recipient).

The authors state that “these results suggest that HDL suppresses the proliferation of myeloid progenitor cells by promoting cholesterol efflux”. Thus, these transporters (intrinsically) or elevated HDL (extrinsically) can regulate hematopoiesis and atherosclerosis. They also reported that stimulating ABCA1/G1 expression above normal levels using a transcriptional activator (TO901317) increased cholesterol efflux and suppressed myeloid cell proliferation, suggesting a new therapeutic rationale.

Statins, such as atorvastatin (Lipitor) and rosuvastatin (Crestor), were designed and are prescribed to reduce serum cholesterol, which is associated with cardiovascular disease. Surprisingly, statins were also found to be anti-inflammatory. Could these results help explain statins' anti-inflammatory effects?

Laurent Yvan-Charvet, Tamara Pagler, Emmanuel L. Gautier, Serine Avagyan, Read L. Siry, Seongah Han, Carrie L. Welch, Nan Wang, Gwendalyn J. Randolph, Hans W. Snoeck, Alan R. Tall, "ATP-Binding Cassette Transporters and HDL Suppress Hematopoietic Stem Cell Proliferation" Science 25 June 2010: Vol. 328. no. 5986, pp. 1689 - 1693

Rapamycin-induced Stress Stimulates anti-Tuberculosis Immunity

Tuberculosis (Tb) is an intracellular mycobacterium that causes 9 million illnesses and nearly 2 million deaths annually world-wide. One third of humans are chronically infected and about 9 million are infected every year, which a vaccine could prevent.

Stressing cells infected by Tb has been shown to induce autophagy ("self-eating", free review), increase presentation of Tb antigens on the infected cell surface, and stimulate specific T lymphocytes. This group found that rapamycin, a pharmaceutical drug used to suppress immunity after solid organ transplantation, induces autophagy and improves immune recognition of Tb. This figure shows that rapamycin treatment of dendritic cells causes a >10-fold increase in their ability to induce anti-Tb immunity in mice (Fig. 5e). Most of the cell culture experiments aimed at dissecting this effect show a much more modest effect of extremely high doses of rapamycin (1 mM!). Oddly, many of the early figures measure IL-2, a hallmark of the T lymphocyte response, even though rapamycin's mode of action inhibiting IL-2 activity would seem to confound the interpretation of the results.

Could this adjuvant effect be translated into a vaccine strategy?

Jagannath C, Lindsey DR, Dhandayuthapani S, Xu Y, Hunter RL Jr, Eissa NT. Autophagy enhances the efficacy of BCG vaccine by increasing peptide presentation in mouse dendritic cells. Nat Med. 2009 Mar;15(3):267-76.