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

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