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: http://www.jimmunol.org/content/188/11/5612.abstract

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: http://jem.rupress.org/content/209/5/1001.abstract

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 intestinal role of NLRC4

The May issue of Nature Immunology contained an article that has described an intriguing mechanism of tolerance to microbiota without losing the ability to detect invading intestinal pathogens and switching on protection mechanisms when it comes to the defending. According to that report macrophages residing in the colon (but not from the bone marrow) remain hyporesponsive to TLR stimulation provided by commensal microbiota. However, the infection with Salmonella can provoke the same macrophages to process and secrete IL-1β cytokine in the manner that is dependent on NLRC4 inflammasome and caspase -1 activation (I have discussed this publication in one of my previous posts: https://memoryreactivation.wordpress.com/2012/05/22/mononuclear-phagocytes-and-the-intestinal-tolerance). The report I am discussing today is related to the above paper because it systematically analyzes the intestinal role of NLRC4. It turns out that there are substantial differences between consequences of deleting NLRC4 compared to effects of TLR5 deletion in conditions when both strains are presumed to harbor the same microbiota (both NLRC4 and TLR5 recognize flagellin – a dominant immune activator in the gut). These differences can be visible either in the healthy colon or in colitis development but not during Salmonella infection.

The link: http://www.nature.com/mi/journal/v5/n3/abs/mi20128a.html

Authors have shown earlier that the deletion of TLR5 caused severe changes in the interactions between host and intestinal commensals even in the absence of any challenge. Such perturbations can lead to the greater bacterial burden in the gut followed by the increase in pro-inflammatory indicators, colitis and eventually the tendency to develop metabolic syndrome diseases (Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5: Science. 2010; 328:228-31). Deletion of NLRC4, however, does not procure any spontaneously occurring physiological changes.

In addition, NLRC4 knockout mice do not display any major effect when they are given injections with anti-IL-10R antibody (the cytokine IL-10 is very important player in maintaining the intestinal tolerance and ablating IL-10 signaling often results in colitis). In contrast, animals with TLR5 or double TLR5/NLRC4 deletions develop colitis upon such treatment. Another way to inflict colitis is to expose the intestinal epithelium to a chemical called DSS which introduces damages to the gut barrier. Investigators show that NLRC4 knockouts similarly to mice with deleted TLR5 are more sensitive to DSS-driven colitis that the wild type strain.

The paper also examines the response against Salmonella infection in mice with NLRC4, TLR5 and double TLR5/NLRC4 deletions. Authors use two strategies – the first applies the procedure resulting in disease that is similar to infection in humans (by pretreating mice with streptomycin prior to bacterial exposure) and serves to examine early inflammatory events in colon and cecum. The second approach includes low-dose oral infection with several Salmonella strains to assess the effect of analyzed deletions on mice survival.

NLRC4 is involved the early detection of Salmonella, although it seems to work together with TLR5. Data show that the single removal of either NLRC4 or TLR5 does not render mice incapable to mount the inflammatory response to this pathogen. Only the double NLRC4/TLR5 deletion strain does not react to Salmonella. The secretion of IL-1β cytokine is also abolished only in the double deletion animals. Interestingly, all double deletion effects (no inflammation and no IL-1β secretion) are mirrored by MyD88 deletion which ablates the signaling to the majority of TLR receptors and the signaling to cytokines processed by NLRC4 inflammasome – IL-1β and IL-18. Authors use this fact to underscore the notion that flagellin (ligand for TLR5) is indeed the dominant immune activator in the gut. Finally, the protective role of NLRC4 is demonstrated by showing that NLRC4 deletion impairs survival to a flagellate strain of Salmonella. In contrast, no any difference between mice with NLRC4 knockout and wild type strain is seen when animals are infected with aflagellate bacteria.

It is hard to directly compare both papers because they have quite different scopes – the first studies only one colonic cell population whereas the other analyzes the overall immune response in the colon. But I think that they corroborate each other versions because unlike TLR5 NLRC4 looks indeed like the immune receptor which is not involved in dealing with microbiota under normal circumstances. However, it is the TLR5/NLRC4 axis that seems be critical in mounting the efficient defense against intestinal pathogens (because only the double TLR5/NLRC4 makes no response to Salmonella). The first paper shows that intestinal macrophages are hyporesponsive to TLR stimulation but they maintain the constitutive expression of pro-IL-1β (which is somehow diminished in germ-free mice). TLR5 is expressed on the surface of antigen presenting cells but also by intestinal epithelial cells whereas NLRC4 is present exclusively inside intestinal macrophages. Could colonic epithelial cells prime these macrophages to express pro-IL-1β when receiving signals through TLR5 from microbiota?

Carvalho FA, Nalbantoglu I, Aitken JD, Uchiyama R, Su Y, Doho GH, Vijay-Kumar M, & Gewirtz AT (2012). Cytosolic flagellin receptor NLRC4 protects mice against mucosal and systemic challenges. Mucosal immunology, 5 (3), 288-98 PMID: 22318495

Mononuclear phagocytes and the intestinal tolerance

I already wrote a couple of entries about microbiota and the fact that our intestinal commensal bacteria do not stimulate aggressive response from the immune system. Most microorganisms share ligands recognized by innate receptors regardless of whether they are pathogens or symbionts. Therefore the way in which our immune system is viewed to operate – by recognition of conserved molecular patterns by antigen presenting cell populations and ensuing activation of immune response – does not explain well the “microbiota problem”. In fact the question how our body makes a distinction between “attack” vs. “hold on” options at mucosal surfaces still needs unraveling. I have found the report that makes an observation on the subject how intestinal tolerance could be maintained without compromising the need for appropriate response when endangered by infectious organisms. This publication suggests that gut-resident antigen presenting cells may be responsive to the presence of certain pathogen-indicating systems but not to ubiquitously present molecular conserved patterns.

The link: http://www.nature.com/ni/journal/v13/n5/full/ni.2263.html

Authors analyze cytokine release pattern (TNF-α, IL-6 and IL-1β) specific to a population coined as intestinal mononuclear phagocytes (iMPs), which are CD11b+ cells isolated from colonic/cecal lamina propria. Most iMPs bear macrophage marker F4/80. These intestinal antigen presenting cells do not respond by making cytokines to several TLR agonists or commensal bacteria but instead they are able to react to the pathogenic bacterium species – Salmonella. Their cytokine profile is also distinct from bone marrow-derived macrophages as they make only IL-1β but not TNF-α or IL-6. In contrast, bone marrow-derived macrophages produce uniformly TNF-α and IL-6 regardless of provided stimulation (TLR ligands or pathogenic bacterium).

IL-1β response by iMPs does not occur when cells are deficient for NLRC4 (cytosolic Nod-like receptor that forms part of inflammasome complex). It is also absent if Salmonella lacks type 3 secretion system (the apparatus that transports bacterial virulence factors into host cell) or flagellin. Developing this observation investigators provide molecular data that link the cleavage of pro-IL-1β into its active form with inflammasome activity (caspase-1 cleavage). Following the above finding, NLRC4-IL-1β axis is shown to be important for the protection against intestinal pathogenic bacterium in an in vivo model. Experimental infections with Salmonella that approximate human disease (by pretreating mice with streptomycin prior to infection) demonstrate worst survival rates for Nlrc4/ and Il1r/ mutants (however, this effect is strain-dependent as it takes place in BALB/c line but not C57BL/6 strain).

The major conclusion of this publication suggests the existence of a detection network that circumvents TLR signaling and relies on inflammasome activation by features unique to pathogens (like type 3 secretion system). However, I have a question that was not answered in the discussion part. Earlier this year the same group has shown that intestinal macrophages very similar to iMPs  (CD11b+F4/80+CD11c-/low) form the source of IL-1β secretion in response to microbiota (I wrote the entry about that publication few months ago – https://memoryreactivation.wordpress.com/2012/03/11/microbiota-il-1%CE%B2-and-th17/). In the paper I am discussing today it is demonstrated that iMPs do not respond by making IL-1β  to commensal bacteria but can be stimulated only by T3SS-possessing pathogenic bacterium. Could it be caused by anatomical differences as this paper studies colonic/cecal lamina propria population and the former investigates processes in small intestine?

Follow-up note: I have contacted the principal investigator with questions concerning both papers. I received comments confirming that these results were caused by different anatomical locations (small vs. large intestine).

Franchi L, Kamada N, Nakamura Y, Burberry A, Kuffa P, Suzuki S, Shaw MH, Kim YG, & Núñez G (2012). NLRC4-driven production of IL-1β discriminates between pathogenic and commensal bacteria and promotes host intestinal defense. Nature immunology PMID: 22484733

TLR7/8 agonist treatment remodels the monocytic population

For quite some time I was trying to put together a post about TLR signaling just to find it very difficult task. I was never really involved in research on innate immunity and as a result I may have oversimplified view on the recognition of conserved molecular patterns. Therefore, despite many interesting publications that I have read recently it was hard to make a decision what to write about. Finally, I have chosen the report describing systemic and local effects of treatment with distinct TLR agonists that are used as adjuvants to boost the immune response. This report is attractive for me because I can learn from it that beside the variety in molecular patterns that are detected, different cellular locations at which the recognition takes place and several downstream signaling pathways which are used to convey activation signals there is yet another layer of complexity pertinent to the innate immunity – altered qualities of response at the systemic level. I am aware that it is probably obvious for somebody in the field.

The link:

http://bloodjournal.hematologylibrary.org/content/119/9/2044.abstract

Authors employ rhesus macaques to examine how TLR-based adjuvants may predispose the overall immune activation at both systemic level (samples collected from blood) and local level (samples collected from draining lymph nodes).  The study uses agonists to TLR4 (MPL), TLR7/8 (R-848) and TLR9 (CpG-ODN) and documents multiple parameters of ensuing immune response. These parameters include blood neutrophils and PMBC levels as well as kinetics of different monocytes subsets both in the blood and in local lymph nodes. Apart from that investigators check the frequency and activation status of dendritic cells (either of myeloid or plasmacytoid origin) and systemic levels of inflammatory cytokines.  Additionally, the study contains transcriptional signatures derived from PMBC and lymph node cells with genes that belong to several classes like adhesion, chemokines, interferon signature or complement.

The data have quite broad scope and I am not going to discuss every result that authors have obtained. The one particular observation, however, I find quite intriguing. As you can probably guess, TLR7/8 agonist stimulates rapid and transient up-regulation of inflammatory cytokines in the blood (IFN-α, IP-10, IL-6, IFN-γ and IL-1Ra) whereas other agonists have much less pronounced effect. This TLR7/8-specific effect appears to be followed by complete remodeling of blood monocytes subsets. Authors dissect circulating monocytes into three classes: classical (CD14+CDCD16), intermediate (CD14+CD16+) and non-classical (CD14dimCD16++). On TLR7/8 agonist treatment there is dramatic but reversible increase in both intermediate and non-classical subsets which normally represent minority of blood monocytes. Some remodeling (but much less prominent) is also documented for TLR9 agonist while TLR4 agonist mobilizes only the classical population.

Primate CD14dim monocytes display excellent crawling and tissue retention capabilities.  These cells have been suggested to become activated by autoimmune complexes and thus contribute to pathology development in lupus. It is also known that signaling through TLRs recognizing nucleic acids (especially TLR7 signaling since TLR9 may have in fact the protective effect) can be involved in the tolerance breach through variety of mechanisms. This publication shows that the significant remodeling of monocyte population on TLR7/8 agonist treatment is reversible. What is the mechanism responsible for return to monocyte homeostasis after receiving activation signals? May this return ability be disturbed in lupus or other autoimmune diseases?

Kwissa, M., Nakaya, H., Oluoch, H., & Pulendran, B. (2012). Distinct TLR adjuvants differentially stimulate systemic and local innate immune responses in nonhuman primates Blood, 119 (9), 2044-2055 DOI: 10.1182/blood-2011-10-388579