On the wild mice and autoimmunity

Only the relatively small part of mammalian genome is formed by protein-coding sequences. The long stretches between these protein-bearing fragments contain other components which could be either non-transcribed regulatory elements or the variety of non-translated RNAs many of which are also taking part in the regulation of gene expression. This intricate network influences the decision whether a given protein is present in the particular physiological situation and how much of it is available. Therefore the issue how proteins are regulated may in fact be equally or more important than the structural differences in coding parts of the protein in question. The problem of transcriptional regulation that affects the autoimmunity development composes the leading theme of the publication I am discussing today. Like the majority of experimental studies this work uses the cornerstone model organism – the mouse Mus musculus. However, unlike the most it provides also some glimpse into the natural populations of rodents.

The link: http://jem.rupress.org/content/209/12/2307.abstract

The particular problem analyzed by this publication comprises the association between FcγRIIb receptor and the aptitude to develop autoimmune reactions. Authors analyze the populations of wild mice from various part of the globe and find out that the vast majority of them are in the possession of FcγRIIb haplotype that is also present in several laboratory mice known for their propensity to develop the autoimmunity. Such arrangement is of interest because it suggests that in the wild populations such autoimmunity-correlated variant of FcγRIIb may actually be positively selected.

FcγRIIb is the receptor that recognizes the constant portion of an antibody that has switched to the IgG isotype and unlike other receptors that also bind to IgG its ligation on B cells causes the modulation of immune responses. To gain an insight of how FcγRIIb haplotype which is predominantly present in the wild mice may influence the immune response of the classic laboratory strain C57BL/6 (which itself has FcγRIIb haplotype not associated with the autoimmunity) investigators exchange the copies of that receptor by the knock-in approach and study the immune parameters of the resulting strain.

It turns out that the alterations between two haplotypes lie in their transcriptional regulation. In the non-autoimmune setting (as demonstrated by C57B/6 strain) FcγRIIb is upregulated on germinal center B cells whereas the knocked-in variant of this receptor cancels this up-regulation. Additionally, when compared to to C57BL/6 line the novel knock-in strain displays the reduced amount of FcγRIIb on activated B cells, splenic transitional B cells and bone marrow residing pre-B cells. Authors pin down the difference in the DNA sequence that underlies the disparate regulation of two variants and propose the transcription factor which may be responsible for the up-regulation seen when the non-autoimmune haplotype is present. Finally, investigators also show that the two haplotypes in questions differ in qualitative terms that comprise the number of germinal center B cells, affinity maturation and autoimmunity development.

Why do I think that this report is interesting? When you look at the paper conclusions from the broader perspective they seem to confirm the notion that in the natural circumstances the autoimmunity is not a problem. The widespread presence of that particular FcγRIIb haplotype among the wild mice suggests that at least for germinal center B cells the natural selection may have favored the situation where the efficiency of immune reaction is maximized even at the cost of potential collateral damage. Maybe it looks like the obvious notion but it is good to have some hard data that confirm it.

Espeli, M., Clatworthy, M., Bokers, S., Lawlor, K., Cutler, A., Kontgen, F., Lyons, P., & Smith, K. (2012). Analysis of a wild mouse promoter variant reveals a novel role for Fc RIIb in the control of the germinal center and autoimmunity Journal of Experimental Medicine, 209 (12), 2307-2319 DOI: 10.1084/jem.20121752

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

Degradation of chemokines by food-borne bacterium

The diet, previous or ongoing encounters with infectious organisms and parasites as well as bacterial microflora that inhabit the intestinal tract or other mucosal surfaces – all these factors influence the quality of immune responses. To mention just one relevant case – in developing countries people appear to be less affected by autoimmune diseases but in wealthy societies autoimmunity represents the constantly growing problem. This phenomenon may be partially due to the much lower level of contact with parasitic worms in places where higher civilization level is attained. It has been shown that some parasites (Schistosoma mansoni is the best known example) seem to be able to exert the regulatory effect on mammalian immune responses and thereby reduce the risk of inappropriate reactions to self-antigens. The microflora may also be a factor in autoimmunity development. The publication I have found describes details of interactions between probiotic-associated bacterial molecule and pro-inflammatory chemokines many of which are involved in autoimmune diseases.

The link: http://www.cell.com/cell-host-microbe/retrieve/pii/S1931312812000662

This report is a continuation of previous study where it has been shown that a cell surface protein from Lactobacillus casei strain derived from VSL#3 (a probiotic food product used in management of ulcerative colitis) can degrade IP-10 (interferon gamma induced protein 10, known also as CXCL10 – a pleiotropic pro-inflammatory chemokine). Authors use molecular biology techniques to prove that the described earlier protein is a serine protease. The action of this protease is not specific to IP-10 as it degrades a number of other chemokines (CXCL9, CXCL11, CXCL12, CX3CL1 and CCL11). On the other hand several well-known pro-inflammatory agents like RANTES, IL-6, IFNγ and TNF are not affected.

Investigators attempt to translate L.casei-dependent chemokine degradation into the physiological setting. To this end they use TNFΔARE/+ model (mice lacking post-transcriptional regulation of TNF that develop spontaneous inflammatory bowel disease). The intraperitoneal injections of L.casei-conditioned media reduces several pro-inflammatory parameters like ileal IP-10 level, activation of certain signaling pathways and infiltration of ileum by mononuclear cells or T cells. Authors proceed to screening fecal human samples for bacteria displaying similar abilities and identify microflora-derived L.casei strain that also degrades IP-10. This strain and its mutated version with the disrupted copy of gene encoding the protease in question are used to feed mice with intestinal inflammation (the different disease model is used this time – Rag2-/- mice that received IL-10 deficient CD4 T cells). The presence of L.casei with protease reduces cecal inflammation indices like IP-10 level or T cell influx whereas the protease-deficient strain is unable to influence the above parameters.

Such capacity of L.casei – the regulation of immune responses via degradation of potent pro-inflammatory agents has provoked me to hypothesize about the origin of dairy products consumption. The hypothesis I have is a tentative one and I am not sure whether it reflects the true reason-effect relationship. I am also aware that it drifts far away from the data presented in the discussed paper and I do not know if it has not has been already proposed by somebody else. Let me state few facts: (1) L.casei belongs to the bacterial group called LAB (lactic acid bacteria). LAB comprises species that are associated with mammalian mucosal surfaces and food products including milk (they are not limited to milk, however). (2) The human capacity to consume milk and dairy products beyond infancy period dates back to the time of agricultural revolution and the transition from hunter-gatherer lifestyle. Interestingly, genomic data indicate that the enzyme metabolizing milk lactose has undergone huge selection events which are comparable to the selection that in the recent human history affected factors responsible for skin pigmentation.

What was the true reason behind our ability to digest milk as adults? Could it be the presence of food-borne bacteria that were able to regulate mucosal immune responses? The transition to the agriculture had to involve many dramatic changes in human diet (most probably in human microbiota, too) and first farmers tended to be actually less healthy than hunters-gatherers (I need to indicate that I have no specialist knowledge of ethnography and I rely here on several general science books I have read – most notably Pandora’s Seed: Why the Hunter-Gatherer Holds the Key to Our Survival by Spencer Wells). Might the extended period of milk consumption be able to alleviate the stress on human physiology imparted by modifications to the original human lifestyle?

Such line of thinking brings another question. What could be special about the original human microbiome? The remaining hunter-gatherer people are distinctively free from diseases comprising so-called metabolic syndrome (source: The Cambridge Encyclopedia of Hunters and Gatherers). Might it be partially due to specific microbiota they harbour? The only relevant report I could find indicates that the oral microbiome of Batwa pygmies is significantly more diverse than in their agricultural neighbors (High diversity of the saliva microbiome in Batwa Pygmies: PLoS One. 2011; 6(8):e23352). Maybe we can find solutions to ever-increasing burdens of civilization in those vanishing people?

von Schillde MA, Hörmannsperger G, Weiher M, Alpert CA, Hahne H, Bäuerl C, van Huynegem K, Steidler L, Hrncir T, Pérez-Martínez G, Kuster B, Haller D. (2012). Lactocepin Secreted By Lactobacillus Exerts Anti-Inflammatory Effects By Selectively Degrading Proinflammatory Chemokines Cell Host&Microbe DOI: 10.1016/j.chom.2012.02.006

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TLR7/8 agonist treatment remodels the monocytic population

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

The link:

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

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

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

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

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