Borrelia burgdorferi – the master manipulator

Who are the most accomplished immunologists in the world? The title may go to several pathogenic organisms that are apparently able to manipulate immune responses and do it in the way that puzzles many researchers. Bacterium Borrelia burgdorferi (the causative agent of Lyme disease) definitely belongs to the elite club. I have learnt that during infection it does not even try to hide away and assumes distinctively bold tactics as it migrates to the very hub of protective action – the draining lymph node. And there it does not sit quietly either since it can cue B cells to what it looks like the unusual (plus yet unexplained) proliferation which probably hinders the quality of ensuing protective response.

The link:

The discussed paper is a continuation of the report which was published by the same group last year (Lymphoadenopathy during lyme borreliosis is caused by spirochete migration-induced specific B cell activation. PLoS Pathog. 7: e1002066). Since I think it is important to combine the information from both papers I am going to summarize shortly the findings of that first publication before moving on to more recent results. Authors observed that when they infected mice with Borrelia using the natural route (tick’s bite) sick animals displayed the substantial enlargement of lymph nodes that were most adjacent to bite locations. In order to control the actual site of infection (ticks are living animals and they can move freely before starting their blood meal) as well as to avoid the direct use of culture-grown bacteria (which may stimulate the different type of immune response than bacteria from infested ticks) investigators have devised a modified infection procedure. Shortly, they injected immunocompromised mice (SCID) with culture-grown Borrelia and transplanted biopsies from such infected animals into the right tarsal joint of naive mice. This innovation has allowed focusing on the single draining lymph node while it exposed animals to host-adapted bacteria.

The particular problem that authors have tackled was how the Borrelia infection altered the right inguinal lymph node and whether there were any further modifications to the lymphatic architecture as the disease progressed. Investigators confirmed the rapid and intense accumulation of B cells in the draining lymph node and also noticed that this accumulation subsequently spread to more distant lymph nodes but not to the spleen. Such ensuing B cell response was critically dependent on the presence of live bacteria inside the lymph node yet quite surprisingly it occurred without any perturbations in the absence of MyD88. Apart from that, authors demonstrated that the immune reaction going on in affected lymph nodes was at least partially specific to Borrelia antigens.

In the follow-up paper researchers attempt to answer the question what is the role of CD4 T cells in the B cell accumulation prompted by Borrelia infection. They find out that CD4 T cells from affected lymph nodes do not increase their numbers as it happens to B cells yet they become activated along the course of disease. Nevertheless, the B cell buildup takes place without CD4 T cells as it did without MyD88. The anti-Borrelia antibody response, however, is weaker when there are no CD4 T cells around.

The overall picture of the immune response to Borrelia in the model that uses host-adapted bacteria (which mimics the natural infection) looks somehow paradoxical and misshapen. First pathogens invade the closest lymph node and seem to provoke there the massive B cell proliferation which disperses later to other lymph nodes. This proliferation is independent of mitogenic cues imparted by TLR signaling and it happens without CD4 T cell-driven costimulation as well. The specific anti-Borrelia antibody response (partially dependent on CD4 T cells) is then switched on but it gives the impression of being not completely normal, too. Authors show that the germinal center induction in lymph nodes is delayed and all germinal centers tend to decline very rapidly. However, plasma cells (which are thought to derive from the germinal center reaction) accumulate with kinetics suggesting that they are not generated in germinal centers located in lymph nodes. Investigators postulate that these plasma cells may originate from ectopic lymphoid tissues.

But it is the initial B cell accumulation that probably distorts the quality of anti-Borrelia immune response. Authors present data showing that this accumulation is indeed able to destroy the inherent organization of an affected lymph node. The question that I have is whether it happens because of sheer number of B cells or maybe through some defined B cell-specific antibody-independent effector mechanism like for example the release of a chemokine that interferes with the layout of a lymph node. Another interesting enigma is how Borrelia targets B cells and what receptor on B cell surface intercepts the signal.

Hastey CJ, Elsner RA, Barthold SW, & Baumgarth N (2012). Delays and diversions mark the development of B cell responses to Borrelia burgdorferi infection. Journal of immunology (Baltimore, Md. : 1950), 188 (11), 5612-22 PMID: 22547698

B cells can secrete IL-6 and drive Th17 response in autoimmunity

The main task of B cells is to release protective immunoglobulins. Yet it is not their only role since they are apparently capable to take on the diverse array of activities that do not directly form a part of effector humoral responses. Instead of just secreting antibodies B cells can influence the outcome of an immune response by dictating the behavior of other cell types. It appears that such mechanism may underlie the development of autoimmunity in the central nervous system. I have found the publication that by combining the work on a mouse experimental model and the analysis of human patients presents the compelling evidence pointing to how B cells stimulate CD4 T cells into the pathogenic phenotype through the antibody-independent action.

The link:

The aim of the paper is to seek a clarification for the unexplained outcomes of various B cell depletion treatments during the course of multiple sclerosis or its mouse model – EAE. For example, the targeted reduction of antibody-secreting plasma cells results in the worsening of disease symptoms. Similarly, the broad B cell depletion leads to the improvement that precedes the drop in the level of autoantibodies. To understand whether B cells may have an additional antibody-independent role in the pathogenesis of multiple sclerosis or EAE investigators focus on IL-6 which is a pro-inflammatory cytokine and the essential factor in autoimmune conditions that afflict the central nervous system. They analyze how B cells can contribute to IL-6 secretion in the context of autoimmunity and assess if B cell-derived IL-6 may influence the conduct of CD4 T cells which are the main factor in multiple sclerosis/EAE development.

Authors show that B cells have the inherent ability to secrete IL-6 when they are stimulated with ligands engaging innate receptors (LPS or CpG plus anti-CD40 antibody) and attempt to prove that such B cells’ capacity may have the physiological importance.  To this end they demonstrate that B cells collected from mice that received EAE-driving immunizations are enriched for IL-6 mRNA compared to B cells from healthy mice. Additionally, the abrogation of IL-6 expression in B cells blunts the severity of neurological symptoms typical to EAE. The final proof indicating for the antibody-independent role of B cells in murine EAE pathogenesis comes from BCDT (B cell depletion therapy) experiments as such treatment is effective in alleviating the EAE-resulting damage only when B cells are competent to release IL-6. On the other hand, knocking out IL-6 expression in B cells does not influence their capacity to secrete antibodies. Investigators also pinpoint that marginal zone B cells are the subset which is most proficient in IL-6 release.

IL-6 is a cytokine involved in the formation of Th17 subset which is known for its participation in pathogenic responses pertinent to the autoimmunity driven by CD4 T cells. Because EAE stands as a representative such disorder, authors ask whether B cell-derived IL-6 could impact the development of Th17 population in the course of this disease. They demonstrate that it is indeed the case since the ablation of IL-6 expression in B cells is able to diminish the propagation of Th17 response during EAE. Remarkably, the B cell-derived IL-6/Th17 axis is operative regardless of antigen specificity as the Th17 population is also less numerous following immunizations with EAE-irrelevant OVA peptide. In the last part of paper devoted to human patients investigators show that the ability of B cells to control Th17 development through IL-6 release may be conserved across mammalian species.

The information that this report contains is obviously important because of its practical value but it also stimulates to ask broader questions. Why we are equipped with signaling pathways like the one described in the discussed report? To what end the apparently pathogenic (in the context of autoimmunity) B cell-derived IL-6/Th17 axis has evolved? Why B cells are so prone to act as autoimmunity mediators when stimulated with TLR ligands (especially nucleic acid-recognizing ligands)? The case of lupus and now that of multiple sclerosis have provided enough evidence for such ostensibly rebellious nature of B cells. Could potentially pathogenic pathways starting with the recognition of TLR ligands by B cells represent an evolutionary trade-off with the better control of gut commensal bacteria as an asset and the danger of autoimmunity as a liability? As a matter of fact MyD88 signaling in B cells has been shown to take part in accommodating the intestinal microbiota by preventing their systemic spread when colonic injury occurs (B Cell-Intrinsic MyD88 Signaling Prevents the Lethal Dissemination of Commensal Bacteria during Colonic Damage: Immunity. 2012; 36 (2): 228-38).

I have one more remark concerning the data that this paper presents. Authors show that CpG (TLR9 ligand) is more efficient that LPS (TLR4 ligand) in driving IL-6 secretion by B cells. Nucleic acid-recognizing TLRs are unusual because they are hidden from the cell surface to endolysosomal compartments. Apart from that, recent publications by Gregory Barton’s group reveal that TLR9, TLR7 and TLR3 (expressed by a macrophage cell line) require proteolytic processing prior to becoming functional detectors. Such feature is interpreted as an additional safety measure since ligands for these receptors are expressed both by host cells and pathogens. I am not really sure if it makes sense but nobody checked if the similar requirement for proteolytic cleavage exists in B cells.

Barr TA, Shen P, Brown S, Lampropoulou V, Roch T, Lawrie S, Fan B, O’Connor RA, Anderton SM, Bar-Or A, Fillatreau S, & Gray D (2012). B cell depletion therapy ameliorates autoimmune disease through ablation of IL-6-producing B cells. The Journal of experimental medicine, 209 (5), 1001-10 PMID: 22547654

The effect of MyD88 deletion on autoimmunity driven by Foxp3 inactivation

Over last 10 years few subjects in immunology have received more attention than regulatory CD4 T cells called in abbreviation Tregs. Tregs are considered to have the potent suppression activity over adaptive immune responses and their lack may result in the autoimmunity development. The central trait pertinent to Tregs is the expression of the transcription factor Foxp3.  The essential role of Foxp3 in Tregs’ life is underscored by the fact that Foxp3-deficient mice or human patients with mutations in the respective gene acquire massive systemic autoimmunity due to the absence of Tregs and generally do not fare well. The publication I have found adds yet another twist to Tregs and Foxp3 story. It turns out that the concurrent to Foxp3 deletion of MyD88 (the crucial adaptor protein linking the innate recognition of microbial signature patterns to the expression of genes involved in defense mechanisms) imparts effects that are not identical between major environmental surfaces (skin, gastrointestinal tract or lungs) and the systemic compartments.

The link:

Foxp3-deficient mice suffer from the advanced inflammatory skin condition and as a result have grossly increased skin pathology indicators like dryness, loss of hair and bleeding. Apart from that their ears and tails are seriously necrotized. However, animals deleted for both Foxp3 and MyD88 show many substantial improvements. Authors demonstrate that the removal of MyD88 from Foxp3-deficient background diminishes immune infiltration to the epidermis and locally deactivates molecular pathways involved in the amplification of inflammatory signals and cellular trafficking (NF-κB translocation to the nucleus, the expression of ICAM-1 on keratinocytes). Additionally, the skin level of numerous cytokines is reduced in doubly deficient animals compared to mice with the single Foxp3 deletion.

What is remarkable, MyD88 deletion on Foxp3-deficient background has also significant systemic effects because such mice grow to much bigger size than visibly runted Foxp3 single mutants. Therefore investigators analyze the extend of immune infiltration in multiple organs of double Foxp3/MyD88 mutants and find out that they have decreased inflammation scores and the expression of pro-inflammatory cytokines not only in the skin but in the small intestine and lungs as well. However, the alleviating consequences of MyD88 removal are restricted to environmental surfaces as the symptoms characteristic for Foxp3 deletion continue unabated in the liver and the pancreas of Foxp3/MyD88-deficient animals (and are even enhanced in their salivary glands). Moreover, the detailed examination of spleen and lymph nodes (authors indicate that they focus on skin draining lymph nodes and mesenteric lymph nodes) shows that cellular counts, proliferation indicators and the expression of various cytokines are elevated in Foxp3/MyD88-deficient mice compared to animals with Foxp3 deletion.

Such difference between the systemic compartments and environmental surfaces could be explained by several factors. Authors show that introducing MyD88 deletion to Foxp3-deficient background disrupts the chemokine gradient between lymph nodes and effector tissues. They also demonstrate that homing ability of CD4 T cells to lungs is incapacitated and as a consequence lymphocytes may accumulate in draining lymph nodes. Finally, in a series of adoptive transfer experiments it is established that the protective effect of MyD88 deletion acts at the level of target tissue and is independent on whether CD4 T cells express MyD88.

In an interesting, although not entirely conclusive part of the paper authors follow the hypothesis that the protective influence of MyD88 deletion in this model may be due to the removal of capability to process activation signals derived from microbiota. To prove such concept they attempt to mimic the effect of MyD88 ablation by purging commensal bacteria from gastrointestinal tracts of Foxp3-deficient animals with two different antibiotic treatments. The first such treatment includes two antibiotics (doxycycline and cotrimoxazole) and indeed relieves some symptoms of Foxp3 inactivation in the skin and lungs. However, the second regimen comprising four antibiotics (kanamycin, vancomycin, metronidizol, and amphotercin-B) actually worsens the state of Foxp3-deficient animals and accelerates their death. I would be interesting to know what part of commensal microflora can be hold responsible for either such protective or detrimental effects in the context of ongoing autoimmunity.

Rivas MN, Koh YT, Chen A, Nguyen A, Lee YH, Lawson G, & Chatila TA (2012). MyD88 is critically involved in immune tolerance breakdown at environmental interfaces of Foxp3-deficient mice. The Journal of clinical investigation, 122 (5), 1933-47 PMID: 22466646

MyD88 signaling in B cells prevents commensals from turning into pathogens

Several years ago Ruslan Medzhitov’s group has published the paper revealing that TLR signaling in response to commensal bacteria is important for the maintenance of intestinal homeostasis. In the model that uses chemical injury (DSS) to compromise the gut integrity such protectiveness provided by recognition of microbiota was completely counter-intuitive result. Why the massive inflammation did not occur when scores of bacterial derivatives have traversed from intestinal lumen to lamina propria and got exposed to the immune system? The recent paper analyses what cellular population is responsible for this unexpected phenomenon and delivers the equally unexpected result – they are B cells. Without MyD88 signaling in B cells commensals that inhabit the gastrointestinal tract show their bad face and turn into pathogens.

The link:

Authors demonstrate that MyD88-deficient mice have compromised survival during DSS treatment and their increased mortality is dependent on the bacterial presence in the gut. The selective inactivation of MyD88 in various cell lineages (epithelial cells, dendritic cells, macrophages, T cells and B cells) shows that the observed mortality effect is due to MyD88 lack in B cells only. The B cell-specific MyD88 signaling seems to regulate IgM response to commensal bacteria. B cells do not need intrinsic MyD88 expression to secrete IgA, although when MyD88 is systemically deleted there is the significant reduction in IgA titers. Finally, the publication points out that IgM-driven complement deposition on microbiota could be accountable for MyD88-dependent homeostasis balance in the gut.

The adaptive response switched on innate-type signals that are received directly by B cells and in which absence commensal microbiota turn to pathogens – such data may have significance that extends beyond strictly practical implications (like new therapies). The intricate relationship between vertebrates and their bacterial symbionts and the impact of microbiota on host physiology are widely accepted facts. But how this association between members of different life domains did originate? I don’t have the answer, nobody probably has and most likely getting such answer is not possible without time machine. However, there are some interesting correlations to be made. Among animals vertebrates and agnathans stand apart as having both the adaptive immune system and the intestinal microflora (at least vertebrates, since I don’t remember reading any paper describing lamprey’s gut bacteria). And despite what an average immunologist trained on mouse models can think of invertebrates – they are highly successful and diverse animals, too. Consider just arthropods or mollusks – they can efficiently defend themselves without the adaptive immunity and spent much more time on this planet than we did. So, what was the evolutionary force behind the formation of adaptive immunity? Maybe it wasn’t protection against infectious organisms?

Kirkland D, Benson A, Mirpuri J, Pifer R, Hou B, Defranco AL, & Yarovinsky F (2012). B Cell-Intrinsic MyD88 Signaling Prevents the Lethal Dissemination of Commensal Bacteria during Colonic Damage. Immunity, 36 (2), 228-38 PMID: 22306056

Microbiota, IL-1β and Th17

After writing my last entry I started deliberating over possible mechanisms that may stay behind the unusual activation status of CD69-positive CD4 T cells that reside in colonic lamina propria. As you remember the presence of commensal microbiota is critical for CD69 up-regulation on CD4 T cells. CD69-positive lymphocytes are also induced into peculiar state of immune unresponsiveness which may be one of mechanisms responsible for intestinal tolerance. Additionally, studies with OVA transgenic strains have shown that colonic CD4 T cells may be able to attain this state in the TCR-independent way. I have found the publication describing microbiota-dependent signaling axis involved in the development of Th17 effector subset in the gut that might be useful for possible elaboration of these findings.

The link:

Th17 is the effector population of CD4 T cells that tends to accumulate inside the gastrointestinal tract in response to commensal microbiota. Furthermore, these cells play the important role in a number of inflammatory and autoimmune conditions. The induction of Th17 in pathogenesis requires several cytokines such as IL-1β, IL-6, IL-23 and TGF-β1. However, it is still not clear which of these factors are necessary for homeostatic intestinal Th17 generation. This publication employs an in vivo reporter system tracking the expression of Th17 lineage-specific transcription factor Ror γt as a marker of Th17 effector population. By analyzing mice deficient in relevant cytokine/cytokine receptors (IL-6, IL-1β and IL-1R) or intracellular signaling (MyD88) authors seek to determine what particular components are necessary for steady-state Th17 intestinal response.

Data indicate that IL1R-MyD88 signaling axis on CD4 T cells is an essential part in developing full-blown Th17 reaction in the gut. According to obtained information MyD88-dependent circuitry can be used twice in the pathway leading to intestinal Th17 generation – first on macrophages that react to conserved molecular patterns derived from microbiota and release IL-1 β; second time on CD4 T cells that bind IL-1 β through IL-1R and activate downstream signaling to become Th17 effector population. In contrast, IL-6 is not needed for steady-state non-inflammatory Th17 level. Authors also identify the macrophage population (CD11b+F4/80+CD11c-/low) as the main source of intestinal IL-1β cytokine.

I wonder how these data might be useful in elaboration of results obtained in CD69-related account. It may be a good idea to check whether the steady activation status of OVA transgenic CD4 T lymphocytes in the colon could be maintained when there is no IL-1R/MyD88 signaling axis on these cells. Other than that this paper is very interesting addition to still developing Th17 story in the gut. For me particularly remarkable is the hint at possible distinction between mechanisms of homeostatic Th17 induction (no IL-6 needed) versus inflammatory Th17 response (with IL-6). It is also not the first suggestion that there may be more than one way to generate IL-17A releasing CD4 T cells – the December issue of Immunity contains the report describing differences in Th17 priming between spleen and gastrointestinal tract. Interestingly, both papers claim that IL-1R/MyD88 signaling axis is important for successful Th17 induction. They disagree, however, about the intestinal role of IL-6 with one paper asserting it dispensable but the other necessary.

What is the homeostatic role of Th17, though? How does it relate to the maintenance of intestinal tolerance? We know quite a lot about Th17 involvement in pathogenesis (here understood either as protection against assaulting bacteria/fungi or aggravation of autoimmune conditions) but much less information is available regarding its steady-state presence. Yet there are some interesting facts to build on. For example, mice are inhabited by the group of microorganisms called segmented filamentous bacteria that can specifically prime Th17 response. These bacteria are non-cultivable and appear to exhibit extreme adaptations for intestinal environment. The gut milieu also seems to be able to enforce the regulatory phenotype on Th17 cells and reduce their pathogenic potential. It is possible that the microbiota-Th17 link may reveal more of its hidden enigmas.

Shaw MH, Kamada N, Kim YG, & Núñez G (2012). Microbiota-induced IL-1β, but not IL-6, is critical for the development of steady-state TH17 cells in the intestine. The Journal of experimental medicine, 209 (2), 251-8 PMID: 22291094