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:

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


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

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

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

The link between Th17 and HIV pathogenesis

The Th17 population is a CD4 T lymphocyte effector subset that in the simplest way can be defined as IL-17A releasing cells; although several other cytokines and transcription factors seem to be typical as well for cells that express IL-17A. These cells are protective in several bacterial and fungal diseases, particularly at mucosal parts of our body. They also are regarded as pathogenic during autoimmune conditions and the infection with Schistosoma mansoni parasite. Another interesting feature of Th17 population is the link to HIV pathogenesis. HIV virus replicates within Th17 subset and IL-17A expressing cells are preferentially depleted from the gut mucosa during HIV infection. The reduction in Th17 level has been linked to increased permeability of mucosal barrier in the digestive tract. This can lead to continuous leaking of microbial products into the bloodstream and maintains chronic immune activation – the HIV infection distinctive feature. Two recent publications reinforce the connection between Th17 and HIV.

The first link:

This publication presents data collected in the region that is affected by AIDS pandemic. Subjects of the study are Kenyan women divided into two pools – virus-negative healthy controls and HIV-positive sex workers. HIV tends to infect activated CD4 T cells that express certain co-receptors facilitating the virus entry. Additionally, α4β7 integrin known to be involved in the mucosal homing binds HIV gp120 envelope protein. Accordingly, authors perform the comparative analysis of cervical and blood CD4 T lymphocytes from healthy donors. This analysis includes enumeration of mucosal homing markers (CCR9, α4β7 and CD103), the early activation marker CD69 and HIV co-receptors (CCR5 and CXCR4).

The research aims to estimate features of potential invasion targets in the female genital tract. As the first conclusion authors have found that cervical CD4 T lymphocytes are enriched for several indicators of HIV susceptibility with α4β7 integrin, CD69 and CCR5 often being co-expressed on individual cells. It has been also established that cervical CD4 T lymphocytes include cells positive for IL-17A. The majority of these IL-17A-positive cells express α4β7 integrin and CCR5 which makes them probable targets during the initial infection stage. Next, authors examine IL-17A expression levels in blood and cervical samples from infected women. The study concludes with the evidence that IL-17A-positive CD4 T cells are strikingly depleted from the cervix mucosa of diseased participants but still present in their blood.

The second link:

Data reported in this article have been obtained from European healthy donors and patients infected with HIV that are treated with anti-retroviral therapy. People infected with HIV are continuously loosing their CD4 T cells and as a result develop the permanent immune deficiency. The anti-retroviral therapy helps rebuilding overall level of CD4 T cells but often proves ineffective in the restoration of intestinal CD4 T cell count. Investigating this matter authors focus on CD4 T lymphocytes from the small intestine and the blood to compare their gut-homing markers expression (α4β7 integrin and CCR9). Healthy controls harbor the vast majority of double positive β7+CCR9+ cells in the gut. However, infected subjects demonstrate the conspicuous reversal in proportions with most of β7+CCR9+ population being present in the blood. Additionally, HIV-positive individuals display the reduction of CCL25 (CCR9 ligand) expression level on intestinal epithelial cells.

This result implies that HIV infection may disturb the redistribution of CD4 T lymphocytes to the gut by unbalancing the CCR9-CCL25 interaction. Authors also associate the low level of intestinal β7+CCR9+ CD4 T cells with increased indicators of damage to the intestinal epithelium. Finally, it is demonstrated that β7+CCR9+ subset is richer in IL-17A secreting cells than β7+CCR9 population. However, in most of their experiments authors did not discriminate between Th17 and other CD4 T cell subsets.

I am following the Th17-HIV story because it emphasizes certain point, which I think is important from the immunologist perspective. Data seem to indicate that HIV infection drives the reduction of Th17 cells at mucosal surfaces. This reduction seems to be irreversible and it disturbs the intestinal homeostasis. Changes in mucosal environment may lead to the enhanced rate of microbial translocation which fuels the unique HIV trait – systemic immune activation. The link between Th17 and HIV pathogenesis makes me wonder whether the consequences of Th17 depletion from mucosal sites arise because of insufficient protection versus enteric pathogens or inadequate accommodation of intestinal commensals.

Our immune system is often metaphorically viewed as well trained and properly equipped coercive body that provides protection against foreign invaders. That’s right – the immune system does provide protection and it is very good at it. But such metaphor might be too narrow to adequately reflect what our immune system actually is, especially at mucosal surfaces. After all we are inhabited by diverse commensal organisms to be reckoned with but not to be disposed of.

Obviously our digestive tract needs protection as it is constantly exposed to harmful bacteria and sometimes the distinction between pathogen and commensal is not entirely clear. Additionally, pathogens are equipped with virulence tools like secretion systems that may provide them with possibilities to stand out from the crowd. But if you consider the sheer biomass argument, commensal organisms prevail over pathogens by such enormous margin that their proper accommodation definitely looks like more urgent matter than the efficient protection against few marauders. So returning to the insufficient protection versus inadequate accommodation dilemma – my gut feeling is that the second option might be more correct.