Schistosoma mansoni evacuates its eggs through Peyer’s patches

Schistosomes are parasitic worms that live in the blood and have a complicated life cycle. The sexual form inhabits vertebrates (humans included) whereas the other stages infest fresh water snails. Adult worms tend to chronically infect their host (sometimes for many years) but for the propagation they have to be able to release their eggs to the outer environment. These blood parasites are remarkably invisible to the immune system; however, their eggs are known to induce the immune response. How such ability may connect to the propagation issue is the subject of the recent publication.

The link: http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003063

Authors study how Schistosoma mansoni, which is an endemic human parasite present in many tropical regions of the world, may excrete its eggs through the intestinal barrier (schistosome species vary between utilization of the intestinal and urinary tracts as the evacuation routes for eggs). It looks like to this end the parasite uses the lymphatic structures encountered in the lower portion of the small intestine – Peyer’s patches. The presence of mature schistosome eggs is apparently able to remodel the vasculature around Peyer’s patches and introduce changes to the cellularity of these intestinal lymphatic structures. Moreover and crucially, in the strain of mice that harbors no Peyer’s patches the egg excretion is visibly reduced and more eggs appear to be backwashed into the host tissue where they form granulomas.

For a number of years schistosome eggs have been hyped as the “taming agent” of the over-reacting immune system. It is well known that they possess the modulatory influences over a number of inflammatory conditions that afflict the gastrointestinal tract. I wonder if such an ability to modulate may be due to the described peculiar interaction of schistosome eggs with their hosts’ Peyer’s patches. From the schistosome point of view eggs need to get out. Thus the parasite may have evolved to drive the immune reaction to its eggs because of the necessity to utilize immune structures for the egg excretion.

Joseph D. Turner,Priyanka Narang,Mark C. Coles,Adrian P. Mountford (2012). Blood Flukes Exploit Peyer’s Patch Lymphoid Tissue to Facilitate Transmission from the Mammalian Host PLOS Pathogens

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Memory CD4 T cells and the neonatal gut

I have found a short paper on the potential mechanism of how HIV virus may be transmitted between mother and child. I think it is interesting because it not only provides the information which may be useful for a given pathology but it also poses some questions as to the basic immunology processes. The main theme of the paper is the quest for HIV targets among neonatal CD4 T cells. As it is known the virus tends to infect memory CD4 T cells but these cells are practically absent in the cord blood. Thus authors inspect neonatal CD4 T populations from various anatomical compartments and find that CD4 T cells bearing a memory marker and HIV co-receptor abound at the intestinal mucosa.

The link: http://bloodjournal.hematologylibrary.org/content/120/22/4383.abstract

CD4 T cells collected for this study derive from children born to healthy mothers therefore this report asks only about the potential mechanism of mother to child transmission. Authors follow CD4 T cells that bear also CD45RO (which is a marker of memory state) and CD5 (HIV uses this molecule as a co-receptor to infect an individual cell – only CD5-tropic strains tend to become transmitted form mother to child). The main conclusion of this publication is that the population of CD4+CD45RO+CD5+cells (the potential HIV target according to the current state of knowledge) exists at the neonatal gut mucosa but not in the lymph nodes, spleen or blood. Additionally, around half of this intestinal memory CD4+CD45RO+CD5population appears to be differentiated into Th17 phenotype since these cells express RORγt transcription factor and CCR6.  In an in vitro experiment investigators also show that neonatal CD4 T cells from the gut are more susceptible to HIV infection than CD4 T cells from the lymph nodes or blood.

Based on obtained data authors propose a model of how HIV gets transmitted from mother to child. According to them the virus may take the oral route of transmition by the ingestion of infected body fluids during the delivery or milk shortly afterwards. I lack the clinical knowledge to critically evaluate such proposal. But I have more basic question instead. This paper not only shows the presence of memory CD4 T cells population at the neonatal gut mucosa but it also provides the evidence that these memory cells underwent substantial clonal expansion that must have happened in utero. I would like to know more details on the nature of antigenic challenge that underlies such prenatal activation of the adaptive immune system.

Bunders MJ, van der Loos CM, Klarenbeek PL, van Hamme JL, Boer K, Wilde JC, de Vries N, van Lier RA, Kootstra N, Pals ST, & Kuijpers TW (2012). Memory CD4+CCR5+ T cells are abundantly present in the gut of newborn infants to facilitate mother-to-child transmission of HIV-1. Blood, 120 (22), 4383-90 PMID: 23033270

First prime and then pull – the novel immunization approach

Some areas of our body enjoy a special status as far as the immune reaction is concerned. Anatomical entities like the gut or female genital tract as well as other mucosal surfaces do not support the same extend of protective response compared to many non-mucosal tissues. This exclusion is crucial to avoid the unwanted inflammation in places that are regularly exposed to the outer environment but sometimes it may present a problem when there is the need to elicit the strong protective response at such privileged site. I have found an interesting report which applies the novel vaccination strategy aimed to enhance the protection against herpes simplex virus 2 which being the virus transmitted through the contact with infected body fluids often enters the body through the genital organs. The innovation that this report introduces consists of double treatment (“prime and pull”) which bypasses the restrictive entry of memory T cells into the vaginal mucosa.

The link: http://www.nature.com/nature/journal/v491/n7424/full/nature11522.html

The mentioned “prime and pull” strategy is the subcutaneous immunization with an attenuated strain of HSV-2 (prime) which is followed by the topical application of chemokines CXCL9 and CXCL10 to the vaginal mucosa (pull). Authors follow the localization of CD8 T cells that recognize an epitope within one of HSV-2 glycoproteins and activated CD4 T cells to show that the distal immunization event plus the localized chemokine treatment provokes the significant recruitment of activated lymphocytes to the vagina whereas the immunization alone has much weaker effect. Interestingly, this recruitment is specific to CD4 and CD8 lymphocytes and does not encompass other cell types that express the relevant chemokine receptor CXCR3.

Is the “prime and pull” approach able to provide the longstanding and reliable protection? Data demonstrate that CD8 T cells (but not CD4 T cells) are retained at vaginal mucosa after the primary response period is over. Most importantly the “prime and pull” treatment may be indeed superior in enforcing the better protective immunity to HSV-2 challenge than the immunization alone. Investigators also ask about the mechanism by which the protection is delivered by the “prime and pull” strategy. It appears that this application can prevent the virus from entering the nervous system where HSV-2 propagates past the mucosal stage of infection.

What will be the future of “prime and pull”, though? The pros are obvious – there is the simple method to enhance the mucosal migration of protective lymphocytes without the “ugly face” of immunity which in this case would be the excessive inflammation at the sensitive anatomical location. Authors speculate about the future applications ranging from HIV protection to solid tumors treatment. The method itself may also be developed as in the discussed paper it provides the optimal protection only in conjunction with the adoptive transfer of virus-specific lymphocytes. The “pull” works as well with the endogenous population of CD8 T cells; however, the protection is suboptimal in such scenario. I will follow this story.

Shin H, & Iwasaki A (2012). A vaccine strategy that protects against genital herpes by establishing local memory T cells. Nature, 491 (7424), 463-467 PMID: 23075848

The own versus foreign microbiota

It was long known that the absence of the gut microbiota impairs the full functionality of mammalian immune system. However, it appears that the immune system may require the species-specific microbiota not just any microbiota to develop its proper responses as the recent publication indicates. I think that this report has important implications both for the better understanding of principal immune events as well as for predicted and much expected innovative research applications like the advent of experimental animals with humanized microbiota.

The link: http://www.cell.com/abstract/S0092-8674(12)00629-0

In the course of this research authors colonize germ-free mice (it means – without any microbiota) with intestinal bacteria that are derived from several sources. Two main of these sources are murine or human fecal samples. Additionally, some experimental mice are also provided with rat microbiota. Summarizing the applied methodology, the starting fecal material (murine, human or rat) serves to prepare a respective probe that provides the formerly germ-free mice with commensal bacteria which differ by the species origin.  Using such model the publication answers two outstanding questions. The first analyzes how different species microbiotas are accommodated inside the murine intestinal tract by looking what is the difference between the original colonization sample and the established microbiota. The second attempts to find out what could be the influence of incongruent microbiota (in this case human or rat-derived) for the development of murine immune responses that are known to be affected by commensal bacteria.

Obviously, humans and mice harbor different microbiotas and data obtained by investigators reflect this simple fact as species identification among two different experimental microbiotas (murine or human-derived) reveals quite dissimilar results. But what is really interesting involves how, or maybe rather to what extend human-derived commensal bacteria could be maintained inside the murine gastrointestinal tract. It appears that recipients of human microbiota demonstrate a period of instability to their intestinal bacterial community after which a constant state is achieved. However, the final microbiota of such mice differs remarkably from the original sample. This is not the case for animals that received murine microbiota. Thus human intestinal commensals cannot be maintained in mice in their entirety. Additional and important piece of information is that Firmicutes may contain the bulk of bacterial species that are specific to humans and unstable in mice.

What is even more striking – mice colonized with human microbiota resemble germ-free mice in many immune parameters that are normally influenced by the presence of commensal bacteria. Studying such mice investigators document many changes in the immune structures of the small intestine such as smaller number of T cells in the lamina propria, less αβ CD4 T cells in the intraepithelial compartment, smaller Peyer’s patches and less T cells inside Peyer’s patches. The large intestine of mice with human microbiota is also affected but in quite contrasting way since it holds less γδ T cells in the intraepithelium but there are no other changes. Peripheral immune organs like spleen or brachial lymph nodes seem to be not altered by the change in microbiota origin. As an additional argument for the need of species-specific microbiota in the proper development of mucosal immune responses authors provide germ-free mice with rat-derived microbiota and observe similar disfuntionalities of the intestinal immune system as in the case of human microbiome transfer.

When it comes to the mechanism responsible for the impaired accumulation of lymphocytes at intestinal sites in mice with humanized microbiota it looks like it is the proliferation in Peyer’s patches and mesenteric lymph nodes that may be hold accountable. On the other hand the gut homing ability seems to not be affected by the heterologous microbiota transfer. Among other results that this publication contains the observation that the colonization with different species microbiota causes different bias in T cell effector phenotypes compared to the colonization with homologous microbiota definitely merits the further attention. Authors come to such conclusion after performing the detailed transcriptional analysis of CD4 T cells from lamina propria isolated from mice that were given the transfer of either murine or human commensal bacteria.

The commentary that I would like to make concerns the differential ability of murine or human microbiotas to stimulate the proliferation of CD4 T cells at mucosal sites. CD4 T cells proliferate as the response to the antigen stimulation and since they bear anticipatory receptors (the true cornerstone of adaptive immunity) the obvious question that comes to mind is why the origin of microbiota matters that much. I do not have explanation for this unexpected result and authors also do not offer a definitive answer, although they make some intelligent guesses as to the potential reason why heterologous microbiota fail to stimulate CD4 T cells proliferation to the same extend as murine commensals (impaired antigen uptake, decreased ability to penetrate mucus layer). An interesting observation is that the host epithelium seems to be more proficient at detecting host-specific than foreign bacteria. Perhaps the stratification by mucus layer is not as stringent in the case of certain host-specific bacterial species.

My last remark touches more practical thing. I have read recently a number of eloquently written review articles that postulated the need to engineer experimental mice with humanized microbiota as the exciting models to study microbiota-influenced diseases like inflammatory bowel disease or metabolic syndrome. But in the light of data that this publication presents the generation of such models looks more complicated than it was thought before. Obviously it is pertinent now that these findings be revisited by other laboratories. The following studies may confirm, widen or even contradict the conclusion presented in the discussed paper. However, maybe we assumed just too much and did not take into account the deep symbiotic relationship between host and its specific commensal bacteria that may be very difficult to recapitulate in a heterologous model.

Chung H, Pamp SJ, Hill JA, Surana NK, Edelman SM, Troy EB, Reading NC, Villablanca EJ, Wang S, Mora JR, Umesaki Y, Mathis D, Benoist C, Relman DA, & Kasper DL (2012). Gut immune maturation depends on colonization with a host-specific microbiota. Cell, 149 (7), 1578-93 PMID: 22726443

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

The developmental diversion of thymocytes

I like reports that make me learn something new and appreciate novel developments leading to more integral view of immunological concepts. My understanding of the thymic central tolerance process was that thymocytes receiving strong signals from tissue antigens through TCR undergo invariably the clonal deletion. But it looks like the clonal deletion of cells that can develop into potentially autoreactive T lymphocytes is not the only way which exists for such population in the thymus. I have read the paper that suggests that there may be actually two outcomes for thymocytes with self-reactive TCR – first is the clonal deletion whereas second the developmental diversion.

The link: http://www.nature.com/ni/journal/v13/n6/abs/ni.2292.html

What is the developmental diversion, though? According to authors it is a process that happens when a thymocyte gets the signal through its autoreactive TCR but is not able to receive the costimulation with CD28 molecule. In such case it can enter a pool of DN cells (double negative for CD4 and CD8) and turn up in the intestinal epithelium where it re-expresses CD8 (in its αα form). Cells derived from the developmental diversion are anergic and when the clonal deletion is impaired (as for example in CD28 knockout mice) the efficiency of central tolerance is not reduced because autoreactive thymocytes have an substitute pathway that sequesters them from harmful and self-reactive mature population.

How the the developmental diversion was detected? The publication contains a lot of data, so I will focus on most crucial evidence. The initial observation made by investigators was that CD28 knockout mice (and also B7 double knockout with no CD80 and CD86 which are CD28 ligands) has unusually numerous population of DN thymocytes that express TCRαβ. In normal mice DN thymocytes are in their majority TCRαβ-negative. The DN population from mice deleted for CD28 contains also the similar proportion of autoreactive TCRs as pre-selection DP (double positive) thymocytes but mature C4 or CD8 T cells from the same strain are mostly deprived of self-reactive rearrangement. Therefore authors conclude that the clonal deletion of autoreactive thymocytes requires CD28 costimulation and in its absence such cells are diverted into the alternative developmental way.

Investigators follow this phenomenon by studying at what exact stage of thymocyte development the diversion may occur and what happens with diverted thymocytes once they leave the thymus (they end up in the intestinal epithelium as already has been remarked). The most interesting thing, however, is that the developmental diversion seems to take place in normal mice as well as TCRαβ+CD8ααintraepithelial lymphocytes from the wild type strain are enriched for autoreactive specificities. I definitely need to start following this story.

Pobezinsky LA, Angelov GS, Tai X, Jeurling S, Van Laethem F, Feigenbaum L, Park JH, & Singer A (2012). Clonal deletion and the fate of autoreactive thymocytes that survive negative selection. Nature immunology, 13 (6), 569-78 PMID: 22544394

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 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: http://www.jci.org/articles/view/40591

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

The early microbiota species composition may influence the B cell development in children

We never walk alone; each of us is colonized by scores of bacterial species that live in our gastrointestinal tract. The colonization event occurs right after birth and has profound effects on our immune system – this sentence appears in every modern immunology textbook. There is, however, no such thing like one human microbiome as the species composition in our gut depends on many aspects. Diet, geographical region we live in or civilization level of society we belong to may influence to what species we are exposed and most probably dictate also many qualitative features of human immune responses. But we do know yet many technical details of the interactions between the human microbiome species arrangement and our immune system characteristics. I have found the publication that studies what impact on the development of B cell populations in the childhood may have the early microbiota species composition in the gut.

The link: http://www.jimmunol.org/content/188/9/4315.abstract

The work is performed on Swedish children and it contains two parts. In the first authors follow the development of B cells in the analyzed cohort of individuals along the period that ranges from right after birth to three years of age. B cells from peripheral blood (defined as CD20+ cells from the lymphocyte gate) are subdivided into CD5+ population (secreting mostly polyreactive IgM antibodies) and CD27+ subset (whose majority are class-switched memory cells). The first population forms the bulk of B cells in children, it expand soon after birth and gradually contracts to reach low level in adults. The second subset grows much slower; it attains its plateau at 18 months of life and continues at similar numbers at 36 months (although there is still much more CD5+ B cells than CD27+ B cells at that time). In the adult blood CD27+ population prevails over CD5+ B cells.

For the second part authors collect fecal samples in the same cohort of children during the early weeks of their life (1, 2, 4 and 8) and study the microbiota species composition in obtained samples. To detect bacterial groups present in the analyzed material they use combined microbiological (growth conditions), biochemical and molecular approaches that identify Escherichia coliEnterobacteria that are not E.coliStaphylococcus aureusBacterioidesEnterococci, BifidobacteriaLactobacilli and Clostridia. Next investigators attempt to correlate quantitative features of B cell development (counts of CD20+, CD5+ and CD27+ cells) along the infancy period with qualitative aspects of microbiota composition (presence or absence of given bacterial groups) during the early weeks of life. The major conclusion of this paper is that the early presence of species or bacterial groups like E.coli and Bifidobacteria in children guts associates with the increased numbers of CD27+ B cells at 4 and 18 months of their life. However, the colonization with S.aureus correlates inversely with CD27+ B cell counts at 4 month.

Obviously correlations do not necessarily indicate for the casual bond but I think the data presented in this report are interesting especially when you combine them with the information that authors provide in the discussion part. According to that E.coli and Bifidobacteria are thought to be classical infantile bacterial species but S.aureus is not. This organism is considered to be a skin bacterium; however, it colonizes the gastrointestinal tract of Swedish children with high penetration (~70%) and may slow down the development of effective immune responses. Such publications are important because the incremental appreciation of how microbiota species composition regulates human immunity may let us use this knowledge for practical purposes in the future.

Lundell AC, Björnsson V, Ljung A, Ceder M, Johansen S, Lindhagen G, Törnhage CJ, Adlerberth I, Wold AE, & Rudin A (2012). Infant B cell memory differentiation and early gut bacterial colonization. Journal of immunology (Baltimore, Md. : 1950), 188 (9), 4315-22 PMID: 22490441

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