A bidirectional association between the gut microbiota and CNS disease in a biphasic murine model of multiple sclerosis.

The gut microbiome plays an important role in the development of inflammatory disease as shown using experimental models of central nervous system (CNS) demyelination. Gut microbes influence the response of regulatory immune cell populations in the gut-associated lymphoid tissue (GALT), which drive protection in acute and chronic experimental autoimmune encephalomyelitis (EAE). Recent observations suggest that communication between the host and the gut microbiome is bidirectional. We hypothesized that the gut microbiota differs between the acute inflammatory and chronic progressive stages of a murine model of secondary-progressive multiple sclerosis (SP-MS). This non-obese diabetic (NOD) model of EAE develops a biphasic pattern of disease that more closely resembles the human condition when transitioning from relapsing-remitting (RR)-MS to SP-MS. We compared the gut microbiome of NOD mice with either mild or severe disease to that of non-immunized control mice. We found that the mice which developed a severe secondary form of EAE harbored a dysbiotic gut microbiome when compared with the healthy control mice. Furthermore, we evaluated whether treatment with a cocktail of broad-spectrum antibiotics would modify the outcome of the progressive stage of EAE in the NOD model. Our results indicated reduced mortality and clinical disease severity in mice treated with antibiotics compared with untreated mice. Our findings support the hypothesis that there are reciprocal effects between experimental CNS inflammatory demyelination and modification of the microbiome providing a foundation for the establishment of early therapeutic interventions targeting the gut microbiome that could potentially limit disease progression.

Influence of the Gut Microbiome on Autoimmunity in the Central Nervous System.

Autoimmune disorders of the CNS have complex pathogeneses that are not well understood. In multiple sclerosis and neuromyelitis optica spectrum disorders, T cells destroy CNS tissue, resulting in severe disabilities. Mounting evidence suggests that reducing inflammation in the CNS may start with modulation of the gut microbiome. The lymphoid tissues of the gut are specialized for the induction of regulatory cells, which are directly responsible for the suppression of CNS-damaging autoreactive T cells. Whether cause or effect, the onset of dysbiosis in the gut of patients with multiple sclerosis and neuromyelitis optica provides evidence of communication along the gut-brain axis. Thus, current and future therapeutic interventions directed at microbiome modulation are of considerable appeal.

Induction of gut regulatory CD39+ T cells by teriflunomide protects against EAE.

OBJECTIVE: To determine whether as an orally delivered treatment, teriflunomide, an inhibitor of the mitochondrial enzyme dihydroorotate dehydrogenase approved to treat relapsing forms of multiple sclerosis, could affect gut-associated lymphoid tissue (GALT) immune responses functionally. METHODS: C57BL/6 mice were treated orally with teriflunomide and flow cytometric analysis of immune GALT cells performed ex vivo, and adoptive transfer experiments were used to test the protective effects of GALT regulatory T (Treg) cells. RESULTS: Teriflunomide reduced the percentages of antigen-presenting cells of Peyer patches when compared to controls. Conversely, a significant increase of the relative frequency of CD39+ Treg cells was observed. In vivo, the protective effect of GALT-derived teriflunomide-induced CD39+ Treg cells was established by adoptive transfer into recipient experimental autoimmune encephalomyelitis mice. CONCLUSIONS: Our results identify specific GALT-derived CD39+ Treg cells as a mechanism of action that may contribute to the efficacy of teriflunomide during CNS inflammatory demyelination and as an oral therapeutic in relapsing multiple sclerosis.

Vitamin A or E and a catechin synergize as vaccine adjuvant to enhance immune responses in mice by induction of early interleukin-15 but not interleukin-1ß responses.

Vitamins A and E and select flavonoids in the family of catechins are well-defined small molecules that, if proven to possess immunomodulatory properties, hold promise as vaccine adjuvants and various therapies. In an effort to determine the in vivo immunomodulatory properties of these molecules, we found that although mucosal and systemic vaccinations with a recombinant HIV-1BaL gp120 with either a catechin, epigallo catechin gallate (EGCG) or pro-vitamin A (retinyl palmitate) alone in a vegetable-oil-in-water emulsion (OWE) suppressed antigen-specific responses, the combination of EGCG and vitamin A or E in OWE (Nutritive Immune-enhancing Delivery System, NIDS) synergistically enhanced adaptive B-cell, and CD4(+) and CD8(+) T-cell responses, following induction of relatively low local and systemic innate tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-17, but relatively high levels of early systemic IL-15 responses. For induction of adaptive interferon-γ and TNF-α responses by CD4(+) and CD8(+) T cells, the adjuvant effect of NIDS was dependent on both IL-15 and its receptor. In addition, the anti-oxidant activity of NIDS correlated positively with higher expression of the superoxide dismutase 1, an enzyme involved in reactive oxygen species elimination but negatively with secretion of IL-1ß. This suggests that the mechanism of action of NIDS is dependent on anti-oxidant activity and IL-15, but independent of IL-1ß and inflammasome formation. These data show that this approach in nutritive vaccine adjuvant design holds promise for the development of potentially safer effective vaccines.

Inflammatory Signals Regulate IL-15 in Response to Lymphodepletion.

Induction of lymphopenia has been exploited therapeutically to improve immune responses to cancer therapies and vaccinations. Whereas IL-15 has well-established roles in stimulating lymphocyte responses after lymphodepletion, the mechanisms regulating these IL-15 responses are unclear. We report that cell surface IL-15 expression is upregulated during lymphopenia induced by total body irradiation (TBI), cyclophosphamide, or Thy1 Ab-mediated T cell depletion, as well as in RAG(-/-) mice; interestingly, the cellular profile of surface IL-15 expression is distinct in each model. In contrast, soluble IL-15 (sIL-15) complexes are upregulated only after TBI or αThy1 Ab. Analysis of cell-specific IL-15Rα conditional knockout mice revealed that macrophages and dendritic cells are important sources of sIL-15 complexes after TBI but provide minimal contribution in response to Thy1 Ab treatment. Unlike with TBI, induction of sIL-15 complexes by αThy1 Ab is sustained and only partially dependent on type I IFNs. The stimulator of IFN genes pathway was discovered to be a potent inducer of sIL-15 complexes and was required for optimal production of sIL-15 complexes in response to Ab-mediated T cell depletion and TBI, suggesting products of cell death drive production of sIL-15 complexes after lymphodepletion. Lastly, we provide evidence that IL-15 induced by inflammatory signals in response to lymphodepletion drives lymphocyte responses, as memory CD8 T cells proliferated in an IL-15-dependent manner. Overall, these studies demonstrate that the form in which IL-15 is expressed, its kinetics and cellular sources, and the inflammatory signals involved are differentially dictated by the manner in which lymphopenia is induced.

The Nlrp3 Inflammasome Suppresses Colorectal Cancer Metastatic Growth in the Liver by Promoting Natural Killer Cell Tumoricidal Activity.

The crosstalk between inflammation and tumorigenesis is now clearly established. However, how inflammation is elicited in the metastatic environment and the corresponding contribution of innate immunity pathways in suppressing tumor growth at secondary sites are poorly understood. Here, we show that mice deficient in Nlrp3 inflammasome components had exacerbated liver colorectal cancer metastatic growth, which was mediated by impaired interleukin-18 (IL-18) signaling. Control of tumor growth was independent of differential cancer cell colonization or proliferation, intestinal microbiota effects, or tumoricidal activity by the adaptive immune system. Instead, the inflammasome-IL-18 pathway impacted maturation of hepatic NK cells, surface expression of the death ligand FasL, and capacity to kill FasL-sensitive tumors. Our results define a regulatory signaling circuit within the innate immune system linking inflammasome activation to effective NK-cell-mediated tumor attack required to suppress colorectal cancer growth in the liver.

Early Effector CD8 T Cells Display Plasticity in Populating the Short-Lived Effector and Memory-Precursor Pools Following Bacterial or Viral Infection.

Naïve antigen-specific CD8 T cells expand in response to infection and can be phenotypically separated into distinct effector populations, which include memory precursor effector cells (MPECs) and short-lived effector cells (SLECs). In the days before the peak of the T cell response, a third population called early effector cells (EECs) predominate the antigen-specific response. However, the contribution of the EEC population to the CD8 T cell differentiation program during an antimicrobial immune response is not well understood. To test if EEC populations were pre-committed to either an MPEC or SLEC fate, we purified EECs from mice infected with Listeria monocytogenes (LM) or vesicular stomatitis virus (VSV), where the relative frequency of each population is known to be different at the peak of the response. Sorted EECs transferred into uninfected hosts revealed that EECs were pre-programmed to differentiate based on early signals received from the distinct infectious environments. Surprisingly, when these same EECs were transferred early into mismatched infected hosts, the transferred EECs could be diverted from their original fate. These results delineate a model of differentiation where EECs are programmed to form MPECs or SLECs, but remain susceptible to additional inflammatory stimuli that can alter their fate.

IL-15 receptor α signaling constrains the development of IL-17-producing γδ T cells.

The development and homeostasis of γδ T cells is highly dependent on distinct cytokine networks. Here we examine the role of IL-15 and its unique receptor, IL-15Rα, in the development of IL-17-producing γδ (γδ-17) T cells. Phenotypic analysis has shown that CD44(high) γδ-17 cells express IL-15Rα and the common gamma chain (CD132), yet lack the IL-2/15Rß chain (CD122). Surprisingly, we found an enlarged population of γδ-17 cells in the peripheral and mesenteric lymph nodes of adult IL-15Rα KO mice, but not of IL-15 KO mice. The generation of mixed chimeras from neonatal thymocytes indicated that cell-intrinsic IL-15Rα expression was required to limit IL-17 production by γδ T cells. γδ-17 cells also were increased in the peripheral lymph nodes of transgenic knock-in mice, where the IL-15Rα intracellular signaling domain was replaced with the intracellular portion of the IL-2Rα chain (that lacks signaling capacity). Finally, an analysis of neonatal thymi revealed that the CD44(lo/int) precursors of γδ-17 cells, which also expressed IL-15Rα, were increased in newborn mice deficient in IL-15Rα signaling, but not in IL-15 itself. Thus, these findings demonstrate that signaling through IL-15Rα regulates the development of γδ-17 cells early in ontogeny, with long-term effects on their peripheral homeostasis in the adult.

CD11a is essential for normal development of hematopoietic intermediates.

The process of lymphopoiesis begins in the bone marrow (BM) and requires multiple cellular intermediates. For T cell production, lymphoid progenitors exit the BM and home to the thymus where maturation and selection ensue. These processes are dependent on a number of factors, including chemokines and adhesion molecules. Although the ß2 integrin CD11a plays an important role in the migration of lymphocytes to lymph nodes, the role of CD11a in T cell development is largely undefined. Our studies now show that, in CD11a(-/-) mice, thymic cellularity was decreased and early T cell development was partially impaired. Remarkably, CD11a was critical for generation of common lymphoid progenitors (CLPs) and lymphoid-primed multipotent progenitors. However, in intact CD11a(-/-) mice, peripheral B and T cell subsets were only modestly altered, suggesting that compensatory mechanisms were operating. In contrast, competitive BM-reconstitution assays revealed an essential role for CD11a in the generation of thymocytes and mature T and B cells. This defect was linked to the requirement for CD11a in the development of CLPs. Furthermore, our results identified CLPs, and not lymphoid-primed multipotent progenitors, as the requisite CD11a-dependent precursor for lymphocyte development. Thus, these findings established a key role for CD11a in lymphopoiesis.

Transcriptional regulation of IL-15 expression during hematopoiesis.

Dendritic cells (DCs) are the most commonly studied source of the cytokine IL-15. Using an IL-15 reporter transgenic mouse, we have recently shown previously unappreciated differences in the levels of IL-15 expressed by subsets of conventional DCs (CD8⁺ and CD8⁻). In this study, we show that IL-15 promoter activity was differentially regulated in subsets of hematopoietically derived cells with IL-15 expression largely limited to myeloid lineages. In contrast, mature cells of the lymphoid lineages expressed little to no IL-15 activity. Surprisingly, we discovered that hematopoietic stem cells (lineage⁻Sca-1⁺c-Kit⁺) expressed high levels of IL-15, suggesting that IL-15 expression was extinguished during lymphoid development. In the case of T cells, this downregulation was Notch-dependent and occurred in a stepwise pattern coincident with increasing maturation and commitment to a T cell fate. Finally, we further demonstrate that IL-15 expression was also controlled throughout DC development, with key regulatory activity of IL-15 production occurring at the pre-DC branch point, leading to the generation of both IL-15⁺CD8⁺ and IL-15(⁻/low)CD8⁻ DC subsets. Thus, IL-15 expression is coordinated with cellular fate in myeloid versus lymphoid immune cells.

Cutting edge: the role of IFN-α receptor and MyD88 signaling in induction of IL-15 expression in vivo.

IL-15 plays a multifaceted role in immune homeostasis, but the unreliability of IL-15 detection has stymied exploration of IL-15 regulation in vivo. To visualize IL-15 expression, we created a transgenic mouse expressing emerald-GFP (EmGFP) under IL-15 promoter control. EmGFP/IL-15 was prevalent in innate cells including dendritic cells (DCs), macrophages, and monocytes. However, DC subsets expressed varying levels of EmGFP/IL-15 with CD8(+) DCs constitutively expressing EmGFP/IL-15 and CD8(-) DCs expressing low EmGFP/IL-15 levels. Virus infection resulted in IL-15 upregulation in both subsets. By crossing the transgenic mice to mice deficient in specific elements of innate signaling, we found a cell-intrinsic dependency of DCs and Ly6C(+) monocytes on IFN-α receptor expression for EmGFP/IL-15 upregulation after vesicular stomatitis virus infection. In contrast, myeloid cells did not require the expression of MyD88 to upregulate EmGFP/IL-15 expression. These findings provide evidence of previously unappreciated regulation of IL-15 expression in myeloid lineages during homeostasis and following infection.

MHC class I and TCR avidity control the CD8 T cell response to IL-15/IL-15Rα complex.

IL-15 operates via a unique mechanism termed transpresentation. In this system, IL-15 produced by one cell type is bound to IL-15Rα expressed by the same cell and is presented to apposing cells expressing the IL-15Rß/γC complex. We have shown that administering soluble IL-15Rα complexed with IL-15 can greatly enhance IL-15 activity. We now show that the naive CD8 T cell response to exogenous IL-15/IL-15Rα complex is MHC class I dependent. In the absence of ß2 microglobulin, naive CD8 T cells scarcely proliferated in response to IL-15/IL-15Rα complex, whereas memory cells proliferated, although to a lesser extent, compared with levels in control mice. The loss of ß2m or FcRn slightly reduced the extended half-life of IL-15/IL-15Rα complex, whereas FcRn deficiency only partially reduced the naive CD8 T cell proliferative response to IL-15/IL-15Rα complex. In addition, we demonstrated a link between TCR avidity and the ability of a T cell to respond to IL-15/IL-15Rα complex. Thus, T cells expressing low-avidity TCR responded poorly to IL-15/IL-15Rα complex, which correlated with a poor homeostatic proliferative response to lymphopenia. The inclusion of cognate peptide along with complex resulted in enhanced proliferation, even when TCR avidity was low. IL-15/IL-15Rα complex treatment, along with peptide immunization, also enhanced activation and the migratory ability of responding T cells. These data suggest that IL-15/IL-15Rα complex has selective effects on Ag-activated CD8 T cells. Our findings have important implications for directing IL-15/IL-15Rα complex-based therapy to specific Ag targets and illustrate the possible adjuvant uses of IL-15/IL-15Rα complex.

The early generation of a heterogeneous CD4+ T cell response to Leishmania major.

CD4(+) T cells are an essential component of both the primary and secondary immune response against the intracellular protozoan parasite Leishmania major. Our laboratory has previously shown that CD62L(high) IL-7R(high) central memory T (T(CM)) cells mediate protective immunity following secondary challenge. To determine when T(CM) cells develop, we examined the phenotype of Leishmania-specific CD4(+) T cells in the first 2 wk following infection. As expected, we identified a population of CD4(+) T cells present in the draining lymph node with the characteristics of effector T cells. However, in addition, a second population phenotypically resembling T(CM) cells emerged coincident with the effector population. These T cells, expressing CD62L, CCR7, and IL-7R, failed to produce IFN-gamma, but had the capacity to give rise to IFN-gamma-producing effector cells. Our studies also demonstrated that the degree of proliferation and the timing of lymph node entry impact T(CM) cell development. The early generation of T(CM) cells following L. major infection indicates that T(CM) cells may not only control secondary infections, but may also contribute to the control of the primary infection.

IL-7 receptor expression provides the potential for long-term survival of both CD62Lhigh central memory T cells and Th1 effector cells during Leishmania major infection.

Infection with the intracellular protozoan parasite Leishmania major induces a state of concomitant immunity wherein secondary immunity is dependent upon the persistence of the original pathogen. Our laboratory has described two populations of Leishmania-induced CD4(+) T cells that contribute to immunity: CD62L(high) central memory T (T(CM)) cells and CD62L(low) effector T cells. To determine whether the prosurvival cytokine IL-7 contributes to maintaining these T cells, we examined expression of the IL7R on CD4(+) T cells activated during L. major infection. We found that T(CM) cells present in chronically infected mice expressed high levels of the IL7R. However, in addition to the expression of the IL7R by T(CM) cells, CD62L(low) cells responding to L. major infection expressed the IL7R. Additional experiments revealed that a large percentage of the IL7R(high)CD62L(low) cells were Th1 cells, based on transcription at the IFN-gamma locus and up-regulation of the Th1-promoting transcription factor T-bet. The up-regulation of T-bet did not prevent IL7R expression by L. major-responding CD4(+) T cells, nor did the absence of T-bet result in increased IL7R expression. Finally, blockade of IL7R signaling decreased the number of T-bet(+)CD4(+) T cells, reduced IFN-gamma production, and inhibited delayed-type hypersensitivity responses in immune mice challenged with L. major, indicating that IL7R signaling contributes to the maintenance of Th1 effector cells. Thus, both T(CM) and Th1 effector cells can express the IL7R during chronic L. major infection, which provides a potential means for their long-term survival in addition to the presence of persisting parasites.

In vivo recognition of ovalbumin expressed by transgenic Leishmania is determined by its subcellular localization.

The importance of the site of Ag localization within microbial pathogens for the effective generation of CD8+ T cells has been studied extensively, generally supporting the view that Ag secretion within infected target cells is required for optimal MHC class I-restricted Ag presentation. In contrast, relatively little is known about the importance of pathogen Ag localization for the activation of MHC class II-restricted CD4+ T cells, despite their clear importance for host protection. We have used the N-terminal targeting sequence of Leishmania major hydrophilic acylated surface protein B to generate stable transgenic lines expressing physiologically relevant levels of full-length OVA on the surface of metacyclic promastigotes and amastigotes. In addition, we have mutated the hydrophilic acylated surface protein B N-terminal acylation sequence to generate control transgenic lines in which OVA expression is restricted to the parasite cytosol. In vitro, splenic dendritic cells are able to present membrane-localized, but not cytosolic, OVA to OVA-specific DO.11 T cells. Strikingly and unexpectedly, surface localization of OVA is also a strict requirement for recognition by OVA-specific T cells (DO.11 and OT-II) and for the development of OVA-specific Ab responses in vivo. However, recognition of cytosolic OVA could be observed with increasing doses of infection. These data suggest that, even under in vivo conditions, where varied pathways of Ag processing are likely to operate, the site of Leishmania Ag localization is an important determinant of immunogenicity and hence an important factor when considering the likely candidacy of vaccine Ags for inducing CD4+ T cell-dependent immunity.

Grating-coupled surface plasmon resonance: a cell and protein microarray platform.

Grating-coupled surface plasmon resonance (GCSPR) is a method for the accurate assessment of analyte in a multiplexed format using small amounts of sample. In GCSPR, the analyte is flowed across specific receptors (e.g. antibodies or other proteins) that have been immobilized on a sensor chip. The chip surface is illuminated with p-polarized light that couples to the gold surface's electrons to form a surface plasmon. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs, thus reducing the intensity of reflected light. Shifts in the GCSPR angle can be correlated with refractive index increases following analyte capture by chip-bound receptors. Because regions of the chip can be independently analyzed, this system can assess 400 interactions between analyte and receptor on a single chip. We have used this label-free system to assess a number of molecules of immunological interest. GCSPR can simultaneously detect an array of cytokines and other proteins using the same chip. Moreover, GCSPR is also compatible with assessments of antigen expression by intact cells, detecting cellular apoptosis and identifying T cells and B cells. This technology represents a powerful new approach to the analysis of cells and molecular constituents of biological samples.