The helminth product ES-62 protects against septic shock via Toll-like receptor 4-dependent autophagosomal degradation of the adaptor MyD88.

Sepsis is one of the most challenging health problems worldwide. Here we found that phagocytes from patients with sepsis had considerable upregulation of Toll-like receptor 4 (TLR4) and TLR2; however, shock-inducing inflammatory responses mediated by these TLRs were inhibited by ES-62, an immunomodulator secreted by the filarial nematode Acanthocheilonema viteae. ES-62 subverted TLR4 signaling to block TLR2- and TLR4-driven inflammatory responses via autophagosome-mediated downregulation of the TLR adaptor-transducer MyD88. In vivo, ES-62 protected mice against endotoxic and polymicrobial septic shock by TLR4-mediated induction of autophagy and was protective even when administered after the induction of sepsis. Given that the treatments for septic shock at present are inadequate, the autophagy-dependent mechanism of action by ES-62 might form the basis for urgently needed therapeutic intervention against this life-threatening condition.

Salmonella SiiE prevents an efficient humoral immune memory by interfering with IgG+ plasma cell persistence in the bone marrow.

Serum IgG, which is mainly generated from IgG-secreting plasma cells in the bone marrow (BM), protects our body against various pathogens. We show here that the protein SiiE of Salmonella is both required and sufficient to prevent an efficient humoral immune memory against the pathogen by selectively reducing the number of IgG-secreting plasma cells in the BM. Attenuated SiiE-deficient Salmonella induces high and lasting titers of specific and protective Salmonella-specific IgG and qualifies as an efficient vaccine against Salmonella A SiiE-derived peptide with homology to laminin ß1 is sufficient to ablate IgG-secreting plasma cells from the BM, identifying laminin ß1 as a component of niches for IgG-secreting plasma cells in the BM, and furthermore, qualifies it as a unique therapeutic option to selectively ablate IgG-secreting plasma cells in autoimmune diseases and multiple myeloma.

Nonfollicular reactivation of bone marrow resident memory CD4 T cells in immune clusters of the bone marrow.

The bone marrow maintains memory CD4 T cells, which provide memory to systemic antigens. Here we demonstrate that memory CD4 T cells are reactivated by antigen in the bone marrow. In a secondary immune response, antigen-specific T cells of the bone marrow mobilize and aggregate in immune clusters together with MHC class II-expressing cells, mostly B lymphocytes. They proliferate vigorously and express effector cytokines, but they do not develop into follicular T-helper cells. Neither do the B lymphocytes develop into germinal center B cells in the bone marrow. Within 10 days, the immune clusters disappear again. Within 30 days, the expanded antigen-specific memory CD4 T cells return to memory niches and are maintained again individually as resting cells. Thus, in secondary immune responses in the bone marrow T-cell memory is amplified, while in germinal center reactions of secondary lymphoid organs humoral memory is adapted by affinity maturation.

CD69+ memory T lymphocytes of the bone marrow and spleen express the signature transcripts of tissue-resident memory T lymphocytes.

It is a matter of current debate whether the bone marrow is a hub for circulating memory T lymphocytes and/or the home of resident memory T lymphocytes. Here we demonstrate for CD69+ murine CD8+ , and CD69+ murine and human CD4+ memory T lymphocytes of the bone marrow, making up between 30 and 60% of bone marrow memory T lymphocytes, that they express the gene expression signature of tissue-resident memory T lymphocytes. This suggests that a substantial proportion of bone marrow memory T lymphocytes are resident. It adds to previous evidence that bone marrow memory T cells are resting in terms of mobility and proliferation, and maintain exclusive long-term memory to distinct, systemic antigens.

Maintenance of CD8+ memory T lymphocytes in the spleen but not in the bone marrow is dependent on proliferation.

It is current belief that numbers of CD8+ memory T lymphocytes in the memory phase of an immune response are maintained by homeostatic proliferation. Here, we compare the proliferation of CD8+ memory T lymphocytes, generated by natural infections and by intentional immunization, in spleen and bone marrow (BM). Fifty percent of CD8+ memory T lymphocytes in the spleen are eliminated by cyclophosphamide within 14 days, indicating that numbers of at least 50% of splenic CD8+ memory T lymphocytes are maintained by proliferation. The numbers of CD8+ memory T lymphocytes in the BM, however, were not affected by cyclophosphamide. This stability was independent of circulating CD8+ memory T cells, blocked by FTY720, showing that BM is a privileged site for the maintenance of memory T lymphocytes, as resident cells, resting in terms of proliferation.

Human memory T cells from the bone marrow are resting and maintain long-lasting systemic memory.

In the bone marrow, a population of memory T cells has been described that promotes efficient secondary immune responses and has been considered to be preactivated, owing to its expression of CD69 and CD25. Here we show that human bone marrow professional memory T cells are not activated but are resting in terms of proliferation, transcription, and mobility. They are in the G0 phase of the cell cycle, and their transcriptome is that of resting T cells. The repertoire of CD4(+) bone marrow memory T cells compared with CD4(+) memory T cells from the blood is significantly enriched for T cells specific for cytomegalovirus-pp65 (immunodominant protein), tetanus toxoid, measles, mumps, and rubella. It is not enriched for vaccinia virus and Candida albicans-MP65 (immunodominant protein), typical pathogens of skin and/or mucosa. CD4(+) memory T cells specific for measles are maintained nearly exclusively in the bone marrow. Thus, CD4(+) memory T cells from the bone marrow provide long-term memory for systemic pathogens.

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 G0 phase of cell cycle and do not express Ki-67. Less than 1% is in S/M/G2 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.

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.

Protection against collagen-induced arthritis in mice afforded by the parasitic worm product, ES-62, is associated with restoration of the levels of interleukin-10-producing B cells and reduced plasma cell infiltration of the joints.

We have previously reported that ES-62, a molecule secreted by the parasitic filarial nematode Acanthocheilonema viteae, protects mice from developing collagen-induced arthritis (CIA). Together with increasing evidence that worm infection may protect against autoimmune conditions, this raises the possibility that ES-62 may have therapeutic potential in rheumatoid arthritis and hence, it is important to fully understand its mechanism of action. To this end, we have established to date that ES-62 protection in CIA is associated with suppressed T helper type 1 (Th1)/Th17 responses, reduced collagen-specific IgG2a antibodies and increased interleukin-10 (IL-10) production by splenocytes. IL-10-producing regulatory B cells have been proposed to suppress pathogenic Th1/Th17 responses in CIA: interestingly therefore, although the levels of IL-10-producing B cells were decreased in the spleens of mice with CIA, ES-62 was found to restore these to the levels found in naive mice. In addition, exposure to ES-62 decreased effector B-cell, particularly plasma cell, infiltration of the joints, and such infiltrating B cells showed dramatically reduced levels of Toll-like receptor 4 and the activation markers, CD80 and CD86. Collectively, this induction of hyporesponsiveness of effector B-cell responses, in the context of the resetting of the levels of IL-10-producing B cells, is suggestive of a modulation of the balance between effector and regulatory B-cell responses that may contribute to ES-62-mediated suppression of CIA-associated inflammation and inhibition of production of pathogenic collagen-specific IgG2a antibodies.

The parasitic helminth product ES-62 suppresses pathogenesis in collagen-induced arthritis by targeting the interleukin-17-producing cellular network at multiple sites.

OBJECTIVE: Among many survival strategies, parasitic worms secrete molecules that modulate host immune responses. One such product, ES-62, is protective against collagen-induced arthritis (CIA), a model of rheumatoid arthritis (RA). Since interleukin-17 (IL-17) has been reported to play a pathogenic role in the development of RA, this study was undertaken to investigate whether targeting of IL-17 may explain the protection against CIA afforded by ES-62. METHODS: DBA/1 mice progressively display arthritis following immunization with type II collagen. The protective effects of ES-62 were assessed by determination of cytokine levels, flow cytometric analysis of relevant cell populations, and in situ analysis of joint inflammation in mice with CIA. RESULTS: ES-62 was found to down-regulate IL-17 responses in mice with CIA. First, it acted to inhibit priming and polarization of IL-17 responses by targeting a complex IL-17-producing network, involving signaling between dendritic cells and γ/δ or CD4+ T cells. In addition, ES-62 directly targeted Th17 cells by down-regulating myeloid differentiation factor 88 expression to suppress responses mediated by IL-1 and Toll-like receptor ligands. Moreover, ES-62 modulated the migration of γ/δ T cells and this was reflected by direct suppression of CD44 up-regulation and, as evidenced by in situ analysis, dramatically reduced levels of IL-17-producing cells, including lymphocytes, infiltrating the joint. Finally, there was strong suppression of IL-17 production by cells resident in the joint, such as osteoclasts within the bone areas. CONCLUSION: Our findings indicate that ES-62 treatment of mice with CIA leads to unique multisite manipulation of the initiation and effector phases of the IL-17 inflammatory network. ES-62 could be exploited in the development of novel therapeutics for RA.

Homeostasis and Durability of T-Cell Memory-The Resting and the Restless T-Cell Memory.

The molecular basis of the persistence of experienced T lymphocytes, also known as "memory T lymphocytes," is still enigmatic. We are beginning to understand their considerable heterogeneity and topographic compartmentalization into memory T cells circulating through the body and those residing in a particular tissue. In some tissues, like murine spleen, subpopulations of memory T cells proliferating in the absence of antigen (homeostatic proliferation) have been described. Other populations are maintained resting in terms of transcription, mobility, and proliferation in dedicated survival niches organized by stromal cells. The survival of these memory T cells is conditional on being in such a niche, where they can persist for a lifetime. Circulating memory T lymphocytes of distinct immune responses slowly decline in numbers over time. The rules governing their entry into and exit from blood, as well as their lifestyle outside of the blood and their relation to resident memory T cells are poorly understood. Homeostasis of circulating, proliferating, and resting memory T cells is obviously controlled by different rheostats: tissue-exit and tissue-entry signals for circulating and proliferation-inducing signals for proliferating memory T cells. For tissue-resident, resting memory T cells, it is the availability of their survival niche. Apparently, this mechanism (i.e., the link between memory T cell and stromal cell) is so robust that it provides efficient T-cell memory over a lifetime in tissues such as the bone marrow.

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 whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility.

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.

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.

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.