Non-pathogenic SIV infection and type-I interferon signaling

How monkeys or apes respond to the challenge of lentiviral immunodeficiency viruses varies across different primate species. Some primates like rhesuses are similar to humans because following SIV infection they develop the AIDS-like disease with all the characteristic features of progressive immune destruction. However, there are other species that do not display such aggravated pathology. African sooty mangabeys are the best studied example among these AIDS-refractory animals. Infected sooty mangabeys do not clear the virus but seem to have adapted to live with it. Such infection lasts for life but it is the relatively mild condition without the continuous depletion of memory CD4 T cells and the chronic immune activation that are associated with human or simian AIDS. The current clinical efforts in humans aim at the reduction of damage caused by the infection and slowing down the progression to AIDS. Thus the detailed knowledge of how AIDS-refractory species achieve their status might be instructive and there is the respective research avenue devoted to studying these species. I have found the publication that looks at the role of type-I interferon signaling during the chronic phase of SIV infection in a species that does not progress to AIDS.

The link: http://bloodjournal.hematologylibrary.org/content/119/24/5750.abstract

Authors attempt to clarify the interactions between the presence of the augmented type-I interferon signaling and the immune response in the chronic phase of SIV infection. Their rationale is simple – since the up-regulation of interferon signature genes correlates with HIV/SIV infections that progress to AIDS, so what may happen if artificially boost the expression of these genes during the non-pathogenic SIV infection? To this end they choose several naturally infected sooty mangabeys and subject them to the treatment with type-I IFN agonist which procures strong but transient enhancement in the expression of interferon signature genes.

To obtain the answer to their question investigators focus on how the increased type-I interferon signaling influences several relevant immune parameters. Acquired data are compared to the baseline values that were observed before the onset of treatment (no control group is included in this research due to availability reasons). The studied parameters comprise the range of CD4 T cells depletion (an indicator of the immune system impairment), the activation and proliferation levels of CD4 T cells (indicators of the chronic immune activation) as well as the intensity of anti-SIV CD8 response.

The take-home message from this report is that the administration of type-I interferon agonist does not impact any of immune parameters that were tested but it only brings down temporarily the viremia level (after all, type-I IFN is regarded as the anti-virus defensive molecule). What does it mean for the understanding of non-pathogenic SIV infection? The mechanisms responsible for the AIDS-refractory status are most probably complex, robust and might not depend on just one particular pathway.

Vanderford TH, Slichter C, Rogers KA, Lawson BO, Obaede R, Else J, Villinger F, Bosinger SE, & Silvestri G (2012). Treatment of SIV-infected sooty mangabeys with a type-I IFN agonist results in decreased virus replication without inducing hyperimmune activation. Blood, 119 (24), 5750-7 PMID: 22550346

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Enter the mycobiota

I have found the publication that focus on pretty much unexplored subject which is the presence and role of commensal fungi in the mammalian gastrointestinal tract. As far as I know there is no information on whether the intestinal fungi community (similarly the bacterial microbiome) has any influence on the basic metabolic functions of their hosts. The discussed paper does not provide such knowledge either. Instead it attempts to establish a link between the increased susceptibility to colitis and the inability to respond properly to fungal wall components (through the lack of the innate receptor Dectin-1) as well as it makes the initial analysis of murine mycobiome. Although it is probably too early to draw such conclusion, my impressions are that there might be differences in the very basics rules of cohabitation between mammals and intestinal fungi compared to mammals/commensal bacteria interactions.

The link: http://www.sciencemag.org/content/336/6086/1314.abstract

Authors confirm the presence of fungi in the gastrointestinal tract with two methods – the first detects the specific fungal RNA whereas the second visualizes fungal cells with soluble Dectin-1 probe (Dectin-1 recognizes β-1,3-glucans from fungal cell wall). The biggest fungal concentration is found in the colon which is also the place where commensal bacteria reach their highest density. However, the bulk of data is devoted to the analysis how the absence of Dectin-1 (which as mentioned above is the fungi-specific innate receptor linked to the inflammasome pathway) may influence the colitis development. The most important finding in that aspect is that the lack of Dectin-1 procures significantly worse colitis outcome in the mouse model that applies DSS-induced injury. Also the polymorphism in human gene encoding Dectin-1 is linked to the severe form of disease recognized as MRUC (medically refractory ulcerative colitis).

The publication contains other interesting data that allow very initial comparison between the characteristics of microbiome and mycobiome. One of important terms that describe a specific interaction between intestinal bacteria and their host is “dysbiosis”. The dysbiosis occurs when the gastrointestinal tract holds the abnormal microflora composition which appears to be able to influence the predisposition to maladies like gut inflammation or metabolic syndrome malfunctions. An interesting example of dysbiosis develops when animals are deficient for the innate receptor that recognizes bacterial flagellin (TLR5) which is a dominant immune activator in the gut. Remarkably, in some cases this pathogenic microflora setup has been shown to be transferable between different specimens as the sheer cohabitation of experimental animals (which is meant to expose them to each other microbiota) may change their susceptibility to certain diseases (consult the following report for an example: Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature; 2012. 482: 179-85). Authors test whether the absence of Dectin-1 could trigger any disease-facilitating microflora variations by crisscross transferring of microflora (not discriminating between bacteria and fungi) from either wild type animals or animals with Dectin-1 deficiency. However, such exchange does not influence the severity of DSS-provoked colitis which in this case looks to be determined by the host genetic background only.

The key in the understanding of the unique interactions between microbiota and immune system is the mutual interdependence of bacteria and their hosts. Nobody knows if this is the case for intestinal fungi; however, the initial data (with the emphasis on “initial”) coming from this report suggest something else. Investigators perform the assessment of murine intestinal mycobiome by sequencing and find that although there is enough diversity in the species arrangement, most data derive from a single organism – Candida tropicalis. This fungus is an opportunistic pathogen and authors confirm that it can play a role in the colitis development. Could intestinal fungi be just free riders?

Iliev ID, Funari VA, Taylor KD, Nguyen Q, Reyes CN, Strom SP, Brown J, Becker CA, Fleshner PR, Dubinsky M, Rotter JI, Wang HL, McGovern DP, Brown GD, & Underhill DM (2012). Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis. Science (New York, N.Y.), 336 (6086), 1314-7 PMID: 22674328

PAX5 ability to repress BLIMP1 is phosphorylation-dependent

When a B cell starts releasing antibodies on the large scale it undergoes the sequence of deep morphological and physiological changes. It expands its cytoplasm and switches on the machinery that lets it cope with the enhanced protein production in preparation to professional antibody secretion. On the molecular level this transition it governed by the onset in expression of certain transcription factors whereas other transcription factors are being switched off. The main players in the transformation of a B cell to a plasma cell are transcription factors PAX5 and BLIMP1. PAX5 is expressed in all B cells except the plasma cell stage while BLIMP1 holds the position of chief regulator that opens the way to antibody secreting cell phenotype. Additionally, it is known that PAX5 represses BLIMP1 and that this repression can be relieved by signaling through BCR. I have read the publication which examines details of the interactions between PAX5, BLIMP1 and the signaling relay activated by BCR stimulation.

The link: http://www.jimmunol.org/content/188/12/6127.abstract

The report’s leading theme is the analysis how the phosphorylation status of PAX5 may influence its ability to repress BLIMP1 promoter. Initially authors identify two residues in PAX5 sequence that can be phosphorylated by ERK kinase which is a part of cascade that transmits the signal originating from BCR. Since the repression competence of PAX5 may be dictated by its phosphorylation state investigators test whether it is possible to activate Blimp-1 promoter when PAX5 is modified to become unresponsive to ERK-driven phosphorylation. To this end they demonstrate that the BCR activation starts off the signaling cascade which terminates with PAX5 phosphorylation by ERK and that the phosphorylated PAX5 is unable to maintain the repression of BLIMP1 promoter. However, the mutated form of PAX5 with its phosphorylation sites replaced by alanine substitutions continues the transcriptional repression of Blimp-1 regardless of BCR activation. Thus the signal at BCR may initiate the plasmacytic differentiation because it changes PAX5 phosphorylation status.

The discussed publication studies the molecular events only and due to a number of technical limitations (see the discussion chapter in the original paper) it does not attempt to translate its findings to the actual immune response (e.g. whether the same PAX5 modifications may influence plasma cell counts). Why do I think it is interesting, though? The answer is I have just read another paper published by Mark Shlomchik’s group (B Cell Receptor Signal Transduction in the GC Is Short-Circuited by High Phosphatase Activity. Science; 2012. 336: 1178-81). That paper examines the extend of BCR signaling during the germinal center reaction and finds it very limited.

According to authors in GC B cells the phosphorylation level of several components belonging to BCR signaling cascade is diminished compared to non-GC B cells. Such reduction is due to the enhanced phosphatase activity which abolishes signals that could be potentially transmitted downstream when BCR is activated during germinal center reaction. Moreover, the specific to B cells inactivation of SHP-1 phosphatase decreases the GC B cell frequency which indicates that the temporal inhibition of BCR signaling may be actually vital for the proper GC maintenance.

The key findings of these two papers (1) The signaling at BCR may open the way to plasma cell phenotype through phosphorylation cascade which completes with PAX5-mediated relieve of BLIMP1 repression. (2) In GC B cells BCR signaling is limited because of high phosphatase activity make a lot sense when combined together. Germinal centers are anatomical sites where B cells undergo antigen-driven affinity maturation and the GC reaction which is terminated too early would compromise the quality of immune response. It looks like the stimulation at BCR may be able to set off the change in the transcription factor architecture (through the phosphorylation-dependent mechanism) that could promote the immediate plasmacytic differentiation. Therefore the temporal modulation of BCR signaling during GC reaction could be crucial because it would ensure enough time to generate high affinity antibody response. Is this the case? Maybe it is.

Yasuda T, Hayakawa F, Kurahashi S, Sugimoto K, Minami Y, Tomita A, & Naoe T (2012). B cell receptor-ERK1/2 signal cancels PAX5-dependent repression of BLIMP1 through PAX5 phosphorylation: a mechanism of antigen-triggering plasma cell differentiation. Journal of immunology (Baltimore, Md. : 1950), 188 (12), 6127-34 PMID: 22593617

The abluminal crawling of neutrophils

During the inflammatory reaction to an invading pathogen neutrophils arrive at the injury site and leave blood vessels to accumulate around foreign particles. Leukocytes exiting the bloodstream have to breach first through endothelial cells before they enter the interstitial space. The publication I am discussing today adds yet another step to this route as it analyzes how neutrophils migrate through the layer of pericytes that coats endothelial cells. Pericytes are cells that form the important structural part of certain types of blood vessels (capillaries, postcapillary venules, and collecting venules); however, their participation in leukocyte passage was previously unstudied. This paper extends the knowledge of neutrophil migration to the inflamed tissue by describing the novel type of movement coined abluminal crawling and exploring many intricacies of neutrophils/pericytes interactions. My experience in the main technique applied by authors – the real-time in vivo imaging – is next to nothing, but I can still admire how they put to use their skills.

The link: http://jem.rupress.org/content/209/6/1219.abstract

Investigators visualize pericytes and neutrophils by generating the mouse strain that has fluorescent proteins expressed in the above cell types. Additionally, endothelial cells are made visible with the in vivo application of specific immuno-staining.  The migration of neutrophils to peripheral tissue is provoked by the injection of potent inflammatory agent (TNF) into the exposed body part of such mice (cremasteric muscle located in the scrotum). Authors observe that after the rapid breach through endothelial cells neutrophils display prolonged association with the pericyte layer combined with the movement along pericyte processes – the already mentioned abluminal crawling. The abluminal crawling ends when a neutrophil leaves into the interstitial space through a gap between pericytes.

How the abluminal crawling is regulated? The efficient neutrophil movement along pericytes depends on the expression of ICAM-1 (on pericytes) as well as Mac-1 and LFA-1 (on neutrophils). The average gap size between pericytes (which is proposed by investigators to be one of decisive factors underlying successful migration) is enlarged by the injection of proinflammatory cytokines TNF and IL-1β and pericytes express receptors recognizing these mediators. Authors also show the intriguing data suggesting that the gap choice by neutrophils is apparently not random as very often a single gap is used by multiple neutrophils. To sum up, this report implicates that pericytes may participate in the immune response by influencing the neutrophil migration into periphery.

Proebstl D, Voisin MB, Woodfin A, Whiteford J, D’Acquisto F, Jones GE, Rowe D, & Nourshargh S (2012). Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo. The Journal of experimental medicine, 209 (6), 1219-34 PMID: 22615129

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