MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

Proinflammatory type 1 T helper (Th1) cells are enriched in inflamed tissues and contribute to the maintenance of chronic inflammation in rheumatic diseases. Here we show that the microRNA- (miR-) 31 is upregulated in murine Th1 cells with a history of repeated reactivation and in memory Th cells isolated from the synovial fluid of patients with rheumatic joint disease. Knock-down of miR-31 resulted in the upregulation of genes associated with cytoskeletal rearrangement and motility and induced the expression of target genes involved in T cell activation, chemokine receptor– and integrin-signaling. Accordingly, inhibition of miR-31 resulted in increased migratory activity of repeatedly activated Th1 cells. The transcription factors T-bet and FOXO1 act as positive and negative regulators of T cell receptor (TCR)–mediated miR-31 expression, respectively. Taken together, our data show that a gene regulatory network involving miR-31, T-bet, and FOXO1 controls the migratory behavior of proinflammatory Th1 cells

Characterisation of the anti-inflammatory potential of ES-62 in autoimmune disease

Filarial nematode infections remain a significant threat of morbidity in the Tropics. Collectively known as filariasis, these helminth infections can result in gross inflammatory pathology of the infected host. However, more commonly, the host remains relatively asymptomatic, exhibiting a somewhat suppressed, non-inflammatory immune response to the parasite, permitting the longevity of infection commonly seen with many helminth infections. In addition to promoting parasite survival, such immunomodulatory actions appear conducive to host health by limiting development of pathological lesions resulting from aggressive, pro-inflammatory responses. This can prove to be advantageous for humans, as although nematode infection can result in severe pathology, the majority of infected people exhibit little evidence of an inflammatory response or overt tissue destruction/disruption commonly associated with autoimmune disorders. Consistent with this, there is increasing evidence supporting an inverse relationship between worm infection and Th1/17-based inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, type-1 diabetes and multiple sclerosis. Moreover, in the developed world, there has been an alarming increase in these inflammatory diseases, coincident with recent improvements in hygiene; a trend not observed in parasite-endemic countries. Therefore, the ”hygiene hypothesis” proposes that these trends are directly associated, by predicting that in the absence of helminth infection, there is a lack of parasite-induced immunoregulation that can result in an over-active immune response in susceptible individuals, resulting in the development of autoimmunity. Filarial nematodes are known to secrete immunomodulatory excretory-secretory products during infection, which act to modulate inflammatory host immune responses and thus, protect the parasite from elimination. ES-62, a phosphorylcholine-containing glycoprotein secreted by the rodent filarial nematode, Acanthocheilonema viteae, has previously been shown to modulate the responses of several innate cells to promote anti-inflammatory immune responses in vitro and in vivo. Indeed, this laboratory demonstrated that ES-62 exhibited anti-inflammatory properties that extended to potential therapeutic action in autoimmune inflammatory diseases such as Rheumatoid Arthritis (RA). This was evidenced by studies demonstrating that ES-62 not only reduced severity of disease in the murine model of collagen induced arthritis (CIA), but also acted to reduce pro-inflammatory cytokine and auto-antibody production from blood and synovial cultures derived from human RA patients. Perhaps rather surprisingly therefore, ES-62 was subsequently found not to offer any protection in some other models of inflammation, including the NOD mouse model of Type 1 Diabetes, the Plasmodium chabaudi model of malaria and the Toxoplasma gondii model of toxoplasma, all of which have been linked with Th1-like pathology. However, given the recent reassessment of CIA as a Th17-, rather than Th1-, mediated disease, it has been hypothesised that ES-62 exhibits therapeutic potential in models, such as CIA, which reflect Th17- rather than Th1 mediated pathology. As the previous CIA/RA studies did not address the role of the Th17 family of pro-inflammatory cytokine mediators, which have been implicated as orchestrating much of the pathology seen in CIA, the core aim of this thesis was to define whether ES-62 was mediating its therapeutic action in autoimmune inflammatory disorders by targeting this Th17 population. Indeed, in chapter 3 of this thesis, it was found that the significant inhibition of disease scores in ES-62-treated CIA mice was accompanied by a significant reduction in levels of IL-17-producing cells, as detected by flow cytometry. Moreover, such analysis revealed that ES-62 reduced IL-17 production from both CD4+ (Th17) cells and γδ cells, which were the two major IL-17-producing populations in the LN of CIA mice. By contrast, ES-62 was not found to modulate the levels of IFNγ-producing cells, either in terms of CD4 or CD8 T cells, which constitute the major cellular compartments generating this cytokine in CIA, supporting the hypothesis that ES-62 targets Th17/IL-17-, rather than Th1-associated inflammation. In addition to suppressing CIA, pilot studies had shown that prophylactic treatment with ES-62 significantly inhibited the development of glomerulonephritis and articular inflammation in MRL/lpr mice, two pathologies commonly associated with SLE in humans. The anti-inflammatory actions of ES-62 were evidenced by a reduction of proteinuria levels and footpad swelling, respectively, but were not associated with modulation of lymphadenopathy, splenomegaly or hypergammaglobulinemia occurring in tandem with autoimmune pathology in such MRL/lpr mice. Studies in chapter 4 of this thesis characterised the cytokine profile of MRL/lpr lupus prone mice, in comparison to congenic MRL/MP control mice, throughout the course of disease induction and progression. These studies showed that IL-17 production was detected in the lymph nodes and serum of MRL/lpr mice, before the onset of lupus pathology. Furthermore, γδ T cells were the major IL-17 producing cell type examined in these mice. Prophylactic treatment with ES-62 resulted in reduced cytokine (IL-17 and IFNγ) release by lymph node cells and splenocytes in response to ex vivo stimulation with the mitogen, ConA, although this did not prove to be significant. As IL-17 was one of the cytokines targeted, this suggested that, as with the CIA model, ES-62 mediated its anti-inflammatory effects through modulation of this cytokine. Consistent with this, it was also found that exposure to ES-62 in vivo reduced the levels of IL-17-producing cells, particularly the γδ T cell population at the earliest time-point examined. By contrast, again as with the CIA model, there was no reduction in the levels of IFNγ-producing cells in ES-62 treated mice, confirming that ES-62 was not acting directly on these Th1 cells. One of the hallmarks of the MRL/lpr model of lupus is the accumulation of B220+CD3+ Double Negative (DN) T cells, which account for much of the lymphadenopathy observed. These cells, which would otherwise have been deleted during development, are known to survive and accumulate due to the lpr (lymphoproliferative) mutation of the Fas gene in these mice. Despite being the predominant LN cell population following onset of disease in MRL/lpr mice, DN T cells have not generally been considered to be pathogenic in this model, but rather proposed to exhibit an anergic-like phenotype. However, some studies have suggested that they can produce cytokines, such as IFNγ and more recently it has been shown that they are a source of IL-17 [1, 2]. Moreover, DN T cells producing IL-17 have been found in the kidneys of SLE patients, suggesting they might indeed play a pathogenic role in lupus-like inflammation [3]. The data presented here established that such DN T cells are capable of producing a range of cytokines, including IL-10, IL-17 and IFNγ and indeed, the most novel finding of these studies was that DN T cells are capable of producing IL-22, a cytokine generally associated with Th17 cells. While the function of this IL-22 production is unclear, it was seen that the percentage of DN T cells producing IL-22 were significantly inhibited by exposure to ES-62 in vivo and hence, whilst ES-62 did not appear to influence the expansion of these cells, it did modulate their effector function by suppressing their capacity to produce this cytokine, although the absolute numbers of cells was not significant reduced. In summary, the novel findings presented in this thesis support the theory that parasite-derived products such as ES-62 may protect against development of the autoimmune inflammatory diseases prevalent in developed society. The highly evolved anti-inflammatory mechanisms designed to beneficially maintain the host-parasite relationship could therefore potentially be exploited to design novel safe therapies for numerous inflammatory diseases. The precise mechanisms by which ES-62 mediates its anti-inflammatory effects have yet to be fully delineated, but it appears to target a variety of cells including IL-17- and IL-22-producing cells (γδ T cells, DN T cells and Th17 cells) in the inflammatory models examined. Importantly, the pilot studies testing the use of synthetic derivatives of ES-62, which showed promise with respect to modulation of inflammatory cytokine production from RA and SLE patient samples, highlight the potential for translating these results into a clinical setting

Laser scanning cytometry: capturing the immune system in situ

Until recently, it has not been possible to image and functionally correlate the key molecular and cellular events underpinning immunity and tolerance in the intact immune system. Certainly, the field has been revolutionized by the advent of tetramers to identify physiologically relevant specificities of T cells, and the introduction of models in which transgenic T-cell receptor and/or B-cell receptor-bearing lymphocytes are adoptively transferred into normal mice and can then be identified by clonotype-specific antibodies using flow cytometry in vitro, or immunohistochemistry ex vivo. However, these approaches do not allow for quantitative analysis of the precise anatomical, phenotypic, signaling, and functional parameters required for dissecting the development of immune responses in health and disease in vivo. Traditionally, assessment of signal transduction pathways has required biochemical or molecular biological analysis of isolated and highly purified subsets of immune system cells. Inevitably, this creates potential artifacts and does not allow identification of the key signaling events for individual cells present in their microenvironment in situ. These difficulties have now been overcome by new methodologies in cell signaling analysis that are sufficiently sensitive to detect signaling events occurring in individual cells in situ and the development of technologies such as laser scanning cytometry that provide the tools to analyze physiologically relevant interactions between molecules and cells of the innate and the adaptive immune system within their natural environmental niche in vivo

The parasitic worm product ES-62 targets myeloid differentiation factor 88-dependent effector mechanisms to suppress antinuclear antibody production and proteinuria in MRL/lpr mice

Objective -- The hygiene hypothesis suggests that parasitic helminths (worms) protect against the development of autoimmune disease via a serendipitous side effect of worm-derived immunomodulators that concomitantly promote parasite survival and limit host pathology. The aim of this study was to investigate whether ES-62, a phosphorylcholine-containing glycoprotein secreted by the filarial nematode Acanthocheilonema viteae, protects against kidney damage in an MRL/lpr mouse model of systemic lupus erythematosus (SLE). Methods -- MRL/lpr mice progressively produce high levels of autoantibodies, and the resultant deposition of immune complexes drives kidney pathology. The effects of ES-62 on disease progression were assessed by measurement of proteinuria, assessment of kidney histology, determination of antinuclear antibody (ANA) production and cytokine levels, and flow cytometric analysis of relevant cellular populations. Results -- ES-62 restored the disrupted balance between effector and regulatory B cells in MRL/lpr mice by inhibiting plasmablast differentiation, with a consequent reduction in ANA production and deposition of immune complexes and C3a in the kidneys. Moreover, by reducing interleukin-22 production, ES-62 may desensitize downstream effector mechanisms in the pathogenesis of kidney disease. Highlighting the therapeutic importance of resetting B cell responses, adoptive transfer of purified splenic B cells from ES-62-treated MRL/lpr mice mimicked the protection afforded by the helminth product. Mechanistically, this reflects down-regulation of myeloid differentiation factor 88 expression by B cells and also kidney cells, resulting in inhibition of pathogenic cross-talk among Toll-like receptor-, C3a-, and immune complex-mediated effector mechanisms. Conclusion -- This study provides the first demonstration of protection against kidney pathology by a parasitic worm-derived immunomodulator in a model of SLE and suggests therapeutic potential for drugs based on the mechanism of action of ES-62

Cutting edge: DNAX accessory molecule 1-deficient CD8+ T cells display immunological synapse defects that impair antitumor immunity

DNAX accessory molecule 1 (DNAM-1) is expressed on all CD8+ T cells and promotes their activation and effector function. DNAM-1 interacts with LFA-1, a critical molecule for immunological synapse formation between T cells and APCs, and for cytotoxic killing of target cells. Mice that lack DNAM-1 display abnormal T cell responses and antitumor activity; however, the mechanism involved is unclear. In this article, we show that DNAM-1 deficiency results in reduced proliferation of CD8+ T cells after Ag presentation and impaired cytotoxic activity. We also demonstrate that DNAM-1-deficient T cells show reduced conjugations with tumor cells and decreased recruitment of both LFA-1 and lipid rafts to the immunological synapse, which correlates with reduced tumor cell killing in vitro. This synapse defect may explain why DNAM-1-deficient mice cannot clear tumors in vivo, and highlights the importance of DNAM-1 and the immunological synapse in T cell-mediated antitumor immunity

Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

At present, it is not clear how memory B lymphocytes are maintained over time, and whetheronly as circulating cells or also residing in particular tissues. Here we describe distinctpopulations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bonemarrow, identified according to individual transcriptional signature and B cell receptorrepertoire. A population of marginal zone-like cells is located exclusively in the spleen, while apopulation of quiescent Bsm is found only in the bone marrow. Three further residentpopulations, present in spleen and bone marrow, represent transitional and follicular B cellsand B1 cells, respectively. A population representing 10-20% of spleen and bone marrowmemory B cells is the only one qualifying as circulating. In the bone marrow, all cells individuallydock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasmacells, are void of activation, proliferation and mobility.Peer Reviewe

Memory CD8+ T cells colocalize with IL-7+ stromal cells in bone marrow and rest in terms of proliferation and transcription

It is believed that memory CD8(+) T cells are maintained in secondary lymphoid tissues, peripheral tissues, and BM by homeostatic proliferation. Their survival has been shown to be dependent on IL-7, but it is unclear where they acquire it. Here we show that in murine BM, memory CD8(+) T cells individually colocalize with IL-7(+) reticular stromal cells. The T cells are resting in terms of global transcription and do not express markers of activation, for example, 4-1BB (CD137), IL-2, or IFN-γ, despite the expression of CD69 on about 30% of the cells. Ninety-five percent of the memory CD8(+) T cells in BM are in G(0) phase of cell cycle and do not express Ki-67. Less than 1% is in S/M/G(2) of cell cycle, according to propidium iodide staining. While previous publications have estimated the extent of proliferation of CD8(+) memory T cells on the basis of BrdU incorporation, we show here that BrdU itself induces proliferation of CD8(+) memory T cells. Taken together, the present results suggest that CD8(+) memory T cells are maintained as resting cells in the BM in dedicated niches with their survival conditional on IL-7 receptor signaling