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:

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

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:

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

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:

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 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:

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

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

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

The link:

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

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

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

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

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

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

The effect of MyD88 deletion on autoimmunity driven by Foxp3 inactivation

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

The link:

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

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

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

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

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

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: 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:

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

Neutrophils, IL-17 and oral microbiota in periodontitis

Th17 response has the ability to induce migration of neutrophils from the bloodstream into inflamed tissues. Neutrophils form the important part of immune defense and as such are equipped with a number of anti-microbial and pro-inflammatory measures. However, as in the case of other immune effectors their action may also have the darker side – collateral injuries to the surrounding environment. Such immune-mediated bystander damages underlie the pathology of periodontitis which is the inflammatory disease afflicting gingival tissue. I have read very interesting publication that describes how the intricate interplay between neutrophils and IL-17 cytokine may regulate periodontitis development.

The link:

Periodontitis is a disease that inflicts neutrophil-mediated inflammatory lesions to the tooth-supportive tissue which may result in loosening and loss of teeth. Old mice just like old humans are prone to develop periodontitis. Del -1 is a negative regulator of neutrophil extravasation which is known to be expressed by endothelial cells (cells that line blood vessels). Authors initially explored Del-1 expression pattern in various age groups as the association between the age and the disposition to excessive inflammation is well known. They concluded that Del-1 level in the gingival tissue of older mice represented only a small portion (~25%) of Del-1 amount found in younger mice. They also found that the decrease in Del-1 expression correlated with more pronounced tooth bone loss and the enhanced neutrophil influx to gingiva. The Del-1/neutrophils/bone loss relationship was confirmed by using Del-1 deficient mice. Apart from that it was shown that the increase in gingival neutrophil infiltration mediated by the deletion of Del-1 could be counteracted by knocking out LFA-1 which is a positive regulator involved in neutrophil tissue migration.

Del-1 deletion seemed to augment the local Th17 response as Del-1 deficient mice expressed more IL-17A (IL-17C and IL-17F were also increased) as well as p40 and p19 (subunits of IL-23 which is strong Th17 response inducer). Remarkably, the analysis of IL-17RA-deficient mice has shown that inflammatory bone loss characteristic for periodontal injuries was completely abolished when there was no IL-17 signaling (interestingly, there was no difference between IL17RA-deficient strain and combined IL-17RA/Del-1 mutant). IL-17RA deletion also enhanced Del-1 expression in gingival tissues. Investigators confirmed the link between lack of IL-17 signaling and increase in Del-1 amount by neutralization method (injection of monoclonal anti-IL17 antibody to gingiva) and bone marrow chimeras experiments. The last approach revealed that the lack of IL-17RA on non-hematopoietic cells was important in regulating Del-1 expression. Finally, authors explored whether administration of Del-1 into inflamed gingival tissues could have the therapeutic potential

Beyond results discussed above this publication contains data on how genetic background that underlies quantitative aspects of neutrophil migration into gingiva may influence the oral microbiota. These data are somewhat counter-intuitive (as it is sometimes the case for things happening at mucosal surfaces). The leading theme of this paper is that Del-1 down-regulation or absence promotes the enhanced neutrophil infiltration and augments inflammatory-mediated tooth bone loss. However, Del-1 deletion (and hence more gingival neutrophils, which after all are thought to be cells with anti-microbial properties) actually stimulates more bacterial growth. This not just one odd result, because when Del-1 deletion is combined with LFA-1 knockout (a positive regulator of neutrophil extravasation) or IL-17RA knockout (removing thus the important part of signaling involved in the neutrophil influx) the number of oral anaerobic bacteria goes back to normal values. Thus to sum up, the excessive inflammation seems to boost but not to restrict the growth of oral microbiota. Authors do not follow this observation but I think it is very interesting phenomenon.

Eskan, M., Jotwani, R., Abe, T., Chmelar, J., Lim, J., Liang, S., Ciero, P., Krauss, J., Li, F., Rauner, M., Hofbauer, L., Choi, E., Chung, K., Hashim, A., Curtis, M., Chavakis, T., & Hajishengallis, G. (2012). The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss Nature Immunology, 13 (5), 465-473 DOI: 10.1038/ni.2260

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:

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 – 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

Plasmacytoid dendritic cells may have the role in central tolerance

The principle of central tolerance implies that developing T cells be exposed to tissue antigens and become deleted if they display auto-reactivity features. However, the understanding of gene expression control has engendered the problem of how peripheral tissue-specific antigens reach the anatomic organ where the central tolerance process takes place – the thymus. Among mechanisms that provide explanation for this problem are those that either circumvent the gene expression aspect (passive diffusion of antigens through the blood) or embrace it (promiscuous intrathymic gene expression). There are also data that indicate the tolerogenic role for dendritic cells residing in thymic compartments. These dendritic cells have been suggested to be able to collect peripheral antigens in tissues and supply them to the thymus. In this line I have found very interesting publication that describes plasmacytoid dendritic cells as potential players in the central tolerance induction.

The link:

Authors show that CCR9-deficient mice have decreased numbers of thymic plasmacytoid dendritic cells. Following this initial finding it is concluded that WT pDCs possess the advantage over CCR9-deficient pDCs in homing ability to the thymus and additionally to the lamina propria and the intestinal intraepithelium. To prove such point investigators have used diverse techniques that included parabiosis experiments (animals of different genetic background that are surgically joined to enable blood cells exchange), generation of bone marrow chimaeras combined with adoptive transfer methodology and in vivo enrichment of dendritic cell populations by grafting mice with tumors that express Flt3L.

To address the possible role of plasmacytoid dendritic cells in the central tolerance authors examine if pDCs may be able to transport OVA antigen to the thymus in order to induce the deletion of OVA-specific CD4 T cells there. Following the generation of bone marrow chimaeras that enable monitoring fate of transgenic OT-II thymocytes and the intravenous injection of pDCs loaded with OVA antigen it is demonstrated that this is indeed the case. However, the tolerogenic activity of pDCs is abrogated when these cells become activated with TLR9 ligand. For the full ability to delete CD4 T cells pDCs need to express CCR9 – as this molecule directs them to the thymus but is not involved in the proper deletion mechanism. The publication also contains clever visualization scheme devised to prove that pDCs are actually capable to collect tissue antigens and shuttle them to the thymus.

So, it seems like plasmacytoid dendritic cells beyond their known participation in viral defense and peripheral Treg induction may have an additional function. The deletion data obtained with OVA transgenic system is very convincing and entice to seek some further evidences supporting this novel physiological role of pDCs. For example, it might be interesting to look for a link between the ability of pDCs to transport peripheral antigens to the thymus and tolerance to intestinal commensal flora. It is well established that postpartum bacterial colonization has profound effects on the functionality of immune system. May it also influence the central tolerance process? I don’t know the answer, but I think there are tools like gnotobiotic mono-colonized mice and TCR transgenic systems that may enable us to get some more information.

Hadeiba H, Lahl K, Edalati A, Oderup C, Habtezion A, Pachynski R, Nguyen L, Ghodsi A, Adler S, & Butcher EC (2012). Plasmacytoid dendritic cells transport peripheral antigens to the thymus to promote central tolerance. Immunity, 36 (3), 438-50 PMID: 22444632