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:

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


MyD88 signaling in B cells prevents commensals from turning into pathogens

Several years ago Ruslan Medzhitov’s group has published the paper revealing that TLR signaling in response to commensal bacteria is important for the maintenance of intestinal homeostasis. In the model that uses chemical injury (DSS) to compromise the gut integrity such protectiveness provided by recognition of microbiota was completely counter-intuitive result. Why the massive inflammation did not occur when scores of bacterial derivatives have traversed from intestinal lumen to lamina propria and got exposed to the immune system? The recent paper analyses what cellular population is responsible for this unexpected phenomenon and delivers the equally unexpected result – they are B cells. Without MyD88 signaling in B cells commensals that inhabit the gastrointestinal tract show their bad face and turn into pathogens.

The link:

Authors demonstrate that MyD88-deficient mice have compromised survival during DSS treatment and their increased mortality is dependent on the bacterial presence in the gut. The selective inactivation of MyD88 in various cell lineages (epithelial cells, dendritic cells, macrophages, T cells and B cells) shows that the observed mortality effect is due to MyD88 lack in B cells only. The B cell-specific MyD88 signaling seems to regulate IgM response to commensal bacteria. B cells do not need intrinsic MyD88 expression to secrete IgA, although when MyD88 is systemically deleted there is the significant reduction in IgA titers. Finally, the publication points out that IgM-driven complement deposition on microbiota could be accountable for MyD88-dependent homeostasis balance in the gut.

The adaptive response switched on innate-type signals that are received directly by B cells and in which absence commensal microbiota turn to pathogens – such data may have significance that extends beyond strictly practical implications (like new therapies). The intricate relationship between vertebrates and their bacterial symbionts and the impact of microbiota on host physiology are widely accepted facts. But how this association between members of different life domains did originate? I don’t have the answer, nobody probably has and most likely getting such answer is not possible without time machine. However, there are some interesting correlations to be made. Among animals vertebrates and agnathans stand apart as having both the adaptive immune system and the intestinal microflora (at least vertebrates, since I don’t remember reading any paper describing lamprey’s gut bacteria). And despite what an average immunologist trained on mouse models can think of invertebrates – they are highly successful and diverse animals, too. Consider just arthropods or mollusks – they can efficiently defend themselves without the adaptive immunity and spent much more time on this planet than we did. So, what was the evolutionary force behind the formation of adaptive immunity? Maybe it wasn’t protection against infectious organisms?

Kirkland D, Benson A, Mirpuri J, Pifer R, Hou B, Defranco AL, & Yarovinsky F (2012). B Cell-Intrinsic MyD88 Signaling Prevents the Lethal Dissemination of Commensal Bacteria during Colonic Damage. Immunity, 36 (2), 228-38 PMID: 22306056

Microbiota, IL-1β and Th17

After writing my last entry I started deliberating over possible mechanisms that may stay behind the unusual activation status of CD69-positive CD4 T cells that reside in colonic lamina propria. As you remember the presence of commensal microbiota is critical for CD69 up-regulation on CD4 T cells. CD69-positive lymphocytes are also induced into peculiar state of immune unresponsiveness which may be one of mechanisms responsible for intestinal tolerance. Additionally, studies with OVA transgenic strains have shown that colonic CD4 T cells may be able to attain this state in the TCR-independent way. I have found the publication describing microbiota-dependent signaling axis involved in the development of Th17 effector subset in the gut that might be useful for possible elaboration of these findings.

The link:

Th17 is the effector population of CD4 T cells that tends to accumulate inside the gastrointestinal tract in response to commensal microbiota. Furthermore, these cells play the important role in a number of inflammatory and autoimmune conditions. The induction of Th17 in pathogenesis requires several cytokines such as IL-1β, IL-6, IL-23 and TGF-β1. However, it is still not clear which of these factors are necessary for homeostatic intestinal Th17 generation. This publication employs an in vivo reporter system tracking the expression of Th17 lineage-specific transcription factor Ror γt as a marker of Th17 effector population. By analyzing mice deficient in relevant cytokine/cytokine receptors (IL-6, IL-1β and IL-1R) or intracellular signaling (MyD88) authors seek to determine what particular components are necessary for steady-state Th17 intestinal response.

Data indicate that IL1R-MyD88 signaling axis on CD4 T cells is an essential part in developing full-blown Th17 reaction in the gut. According to obtained information MyD88-dependent circuitry can be used twice in the pathway leading to intestinal Th17 generation – first on macrophages that react to conserved molecular patterns derived from microbiota and release IL-1 β; second time on CD4 T cells that bind IL-1 β through IL-1R and activate downstream signaling to become Th17 effector population. In contrast, IL-6 is not needed for steady-state non-inflammatory Th17 level. Authors also identify the macrophage population (CD11b+F4/80+CD11c-/low) as the main source of intestinal IL-1β cytokine.

I wonder how these data might be useful in elaboration of results obtained in CD69-related account. It may be a good idea to check whether the steady activation status of OVA transgenic CD4 T lymphocytes in the colon could be maintained when there is no IL-1R/MyD88 signaling axis on these cells. Other than that this paper is very interesting addition to still developing Th17 story in the gut. For me particularly remarkable is the hint at possible distinction between mechanisms of homeostatic Th17 induction (no IL-6 needed) versus inflammatory Th17 response (with IL-6). It is also not the first suggestion that there may be more than one way to generate IL-17A releasing CD4 T cells – the December issue of Immunity contains the report describing differences in Th17 priming between spleen and gastrointestinal tract. Interestingly, both papers claim that IL-1R/MyD88 signaling axis is important for successful Th17 induction. They disagree, however, about the intestinal role of IL-6 with one paper asserting it dispensable but the other necessary.

What is the homeostatic role of Th17, though? How does it relate to the maintenance of intestinal tolerance? We know quite a lot about Th17 involvement in pathogenesis (here understood either as protection against assaulting bacteria/fungi or aggravation of autoimmune conditions) but much less information is available regarding its steady-state presence. Yet there are some interesting facts to build on. For example, mice are inhabited by the group of microorganisms called segmented filamentous bacteria that can specifically prime Th17 response. These bacteria are non-cultivable and appear to exhibit extreme adaptations for intestinal environment. The gut milieu also seems to be able to enforce the regulatory phenotype on Th17 cells and reduce their pathogenic potential. It is possible that the microbiota-Th17 link may reveal more of its hidden enigmas.

Shaw MH, Kamada N, Kim YG, & Núñez G (2012). Microbiota-induced IL-1β, but not IL-6, is critical for the development of steady-state TH17 cells in the intestine. The Journal of experimental medicine, 209 (2), 251-8 PMID: 22291094

CD69 and mucosal tolerance

Soon after birth every one of us is colonized with multiple bacterial species that stay in our intestinal tract throughout the lifetime.  The gut microbiota participate in many vital processes – they support our digestion and metabolism as well as perform important instructive role for the immune system. Yet these commensal organisms express similar conserved molecular patterns as pathogens and are obviously capable to stimulate the innate arm of immunity. They are also equipped with considerable antigenic load (their biomass is many times bigger than pathogens’ biomass) that has to be the target of adaptive anticipatory receptors. However, in normal circumstances the microbiota presence is accommodated without any ill effects. Why they are not actively assaulted by the immune system? In all probability there is no single and easy answer to this question. How the immune system discriminates between commensal and pathogenic bacteria forms one of the most interesting puzzles faced by the immunology. Our body can use unrecognized strategies to ensure the tolerance at its intestinal compartments.

The link:

This publication is based on the original finding that CD69 (the marker of activated lymphocytes) is predominantly up-regulated by CD4 T cells located at mucosal surfaces. Authors provide evidences that CD69-positive cells appear to be in the state of immune impassiveness as they secrete only TGF-β1 – a cytokine involved in modulation of inflammatory reactions. Additionally, CD69 up-regulation on CD4 T cells is able to suppress the expression of stimulatory cytokines IFN-γ, TNF-α and IL-21. The functional role of CD69 expression is assessed with two experimental approaches – inducible colitis (by adoptive transfer of CD45RBhigh CD4 T cells into RAG / animals) and oral tolerance (by feeding and subsequent immunization with model antigen OVA). It is demonstrated that CD69 deficiency on CD4 T cells causes worse colitis outcome and lack of toleration to orally delivered antigen.

In spite of these intriguing data, I am most interested by one observation that has been left almost unexplored throughout the whole paper. Authors use OVA transgenic (with CD4 T cell compartment transgenic in over 90%) and OVA transgenic on RAG / background (having exclusively transgenic CD4 T cells) strains to show that the cognate activation of CD4 T lymphocytes is necessary to induce CD69 up-regulation. Surprisingly, it looks like the requirement for TCR signaling is not absolute and CD4 T cells from specific anatomical site – the colon – can become activated without TCR ligation (given that we assume that CD69 expression is really the reliable marker of CD4 T cell activation). Data indicate that CD69 expression on OVA transgenic CD4 T cells from the colonic lamina propria is refractory to any reduction even in the absence of OVA. Authors argue that type I IFN signaling is important in both up-regulation of CD69 and protectiveness mediated by CD69-positive cells. It cannot explain, however, why CD4 T cells from colon are so persistently activated without the cognate antigen because IFNAR-deficient mice display similar to wild type levels of CD69 expression (and thus activation) on CD4 T cells at all anatomical locations tested.

In the natural setting CD69 expression is relatively modest on lymphocytes from spleen and mesenteric lymph nodes but it reaches the highest level (approximately 50%) on cells from small intestine and colonic lamina propria. CD69 expression on CD4 T cells is also strictly dependent on the presence of bacterial microbiota which is a common feature of many other immune activities connected to the gastrointestinal tract. The colon is inhabited by greatest number of commensal bacteria than any other part of our body. The steady activation status of colonic CD4 T cells combined with the general modulatory behavior of CD69-positive lymphocytes provokes multiple questions. The most important I would ask is whether the presumed TCR-independent activation of CD4 T cells can be confirmed as the physiologically relevant process occurring in the gut. Can we identify receptors or molecules that convey these activation signals? May it be yet another mechanism accounting for intestinal microbiota tolerance?

Radulovic, K., Manta, C., Rossini, V., Holzmann, K., Kestler, H., Wegenka, U., Nakayama, T., & Niess, J. (2012). CD69 Regulates Type I IFN-Induced Tolerogenic Signals to Mucosal CD4 T Cells That Attenuate Their Colitogenic Potential The Journal of Immunology, 188 (4), 2001-2013 DOI: 10.4049/jimmunol.1100765