High Serum Vitamin D Concentrations, Induced via Diet, Trigger Immune and Intestinal Microbiota Alterations Leading to Type 1 Diabetes Protection in NOD Mice.

The hormonally-active form of vitamin D, 1,25-dihydroxyvitamin D3, can modulate both innate and adaptive immunity, through binding to the nuclear vitamin D receptor expressed in most immune cells. A high dose of regular vitamin D protected non-obese diabetic (NOD) mice against type 1 diabetes (T1D), when initiated at birth and given lifelong. However, considerable controversy exists on the level of circulating vitamin D (25-hydroxyvitamin D3, 25(OH)D3) needed to modulate the immune system in autoimmune-prone subjects and protect against T1D onset. Here, we evaluated the impact of two doses of dietary vitamin D supplementation (400 and 800 IU/day), given to female NOD mice from 3 until 25 weeks of age, on disease development, peripheral and gut immune system, gut epithelial barrier function, and gut bacterial taxonomy. Whereas serum 25(OH)D3 concentrations were 2.6- (400 IU/day) and 3.9-fold (800 IU/day) higher with dietary vitamin D supplementation compared to normal chow (NC), only the 800 IU/day vitamin D-supplemented diet delayed and reduced T1D incidence compared to NC. Flow cytometry analyses revealed an increased frequency of FoxP3+ Treg cells in the spleen of mice receiving the 800 IU/day vitamin D-supplemented diet. This vitamin D-induced increase in FoxP3+ Treg cells, also expressing the ecto-5'-nucleotidase CD73, only persisted in the spleen of mice at 25 weeks of age. At this time point, the frequency of IL-10-secreting CD4+ T cells was also increased in all studied immune organs. High-dose vitamin D supplementation was unable to correct gut leakiness nor did it significantly modify the increased gut microbial diversity and richness over time observed in NOD mice receiving NC. Intriguingly, the rise in alpha-diversity during maturation occurred especially in mice not progressing to hyperglycaemia. Principal coordinates analysis identified that both diet and disease status significantly influenced the inter-individual microbiota variation at the genus level. The abundance of the genera Ruminoclostridium_9 and Marvinbryantia gradually increased or decreased, respectively in faecal samples of mice on the 800 IU/day vitamin D-supplemented diet compared to mice on the 400 IU/day vitamin D-supplemented diet or NC, irrespective of disease outcome. In summary, dietary vitamin D reduced T1D incidence in female NOD mice at a dose of 800, but not of 400, IU/day, and was accompanied by an expansion of Treg cells in various lymphoid organs and an altered intestinal microbiota signature.

Effects of repeated infections with non-typeable Haemophilus influenzae on lung in vitamin D deficient and smoking mice.

BACKGROUND: In chronic obstructive pulmonary disease (COPD), exacerbations cause acute inflammatory flare-ups and increase the risk for hospitalization and mortality. Exacerbations are common in all disease stages and are often caused by bacterial infections e.g., non-typeable Heamophilus influenzae (NTHi). Accumulating evidence also associates vitamin D deficiency with the severity of COPD and exacerbation frequency. However, it is still unclear whether vitamin D deficiency when combined with cigarette smoking would worsen and prolong exacerbations caused by repeated infections with the same bacterial strain. METHODS: Vitamin D sufficient (VDS) and deficient (VDD) mice were exposed to nose-only cigarette smoke (CS) for 14 weeks and oropharyngeally instilled with NTHi at week 6, 10 and 14. Three days after the last instillation, mice were assessed for lung function, tissue remodeling, inflammation and immunity. The impact of VDD and CS on inflammatory cells and immunoglobulin (Ig) production was also assessed in non-infected animals while serum Ig production against NTHi and dsDNA was measured in COPD patients before and 1 year after supplementation with Vitamin D3. RESULTS: VDD enhanced NTHi eradication, independently of CS and complete eradication was reflected by decreased anti-NTHi Ig's within the lung. In addition, VDD led to an increase in total lung capacity (TLC), lung compliance (Cchord), MMP12/TIMP1 ratio with a rise in serum Ig titers and anti-dsDNA Ig's. Interestingly, in non-infected animals, VDD exacerbated the CS-induced anti-NTHi Ig's, anti-dsDNA Ig's and inflammatory cells within the lung. In COPD patients, serum Ig production was not affected by vitamin D status but anti-NTHi IgG increased after vitamin D3 supplementation in patients who were Vitamin D insufficient before treatment. CONCLUSION: During repeated infections, VDD facilitated NTHi eradication and resolution of local lung inflammation through production of anti-NTHi Ig, independently of CS whilst it also promoted autoantibodies. In COPD patients, vitamin D supplementation could be protective against NTHi infections in vitamin D insufficient patients. Future research is needed to decipher the determinants of dual effects of VDD on adaptive immunity. TRAIL REGISTRATION: ClinicalTrials, NCT00666367. Registered 23 April 2008, https://www.clinicaltrials.gov/ct2/show/study/NCT00666367 .

Spatial covariation in nutrient enrichment and fishing of herbivores in an oceanic coral reef ecosystem.

Both natural and anthropogenic stressors are increasing on coral reefs, resulting in large-scale loss of coral and potential shifts from coral- to macroalgae-dominated community states. Two factors implicated in shifts to macroalgae are nutrient enrichment and fishing of reef herbivores. Although either of these factors alone could facilitate establishment of macroalgae, reefs may be particularly vulnerable to coral-to-algae phase shifts in which strong bottom-up forcing from nutrient enrichment is accompanied by a weakening of herbivore control of macroalgae via intense fishing. We explored spatial heterogeneity and covariance in these drivers on reefs in the lagoons of Moorea, French Polynesia, where the local fishery heavily targets herbivorous fishes and there are spatially variable inputs of nutrients from agricultural fertilizers and wastewater systems. Spatial patterns of fishing and nutrient enrichment were not correlated at the two landscape scales we examined: among the 11 interconnected lagoons around the island or among major habitats (fringing reef, mid-lagoon, back reef) within a lagoon. This decoupling at the landscape scale resulted from patterns of covariation between enrichment and fishing that differed qualitatively between cross-shore and long-shore directions. At the cross-shore scale, nutrient enrichment declined but fishing increased from shore to the crest of the barrier reef. By contrast, nutrient enrichment and fishing were positively correlated in the long-shore direction, with both increasing with proximity to a pass in the barrier reef. Contrary to widespread assumptions in the scientific literature that human coastal population density correlates with impact on marine ecosystems and that fishing effort declines linearly with distance from the shore, these local stressors produced a complex spatial mosaic of reef vulnerabilities. Our findings support spatially explicit management involving the control of anthropogenic nutrients and strategic reductions in fishing pressure on herbivores by highlighting specific areas to target for management actions.

Peptidylarginine Deiminase Inhibition Prevents Diabetes Development in NOD Mice.

Protein citrullination plays a role in several autoimmune diseases. Its involvement in murine and human type 1 diabetes has recently been recognized through the discovery of antibodies and T-cell reactivity against citrullinated peptides. In the current study, we demonstrate that systemic inhibition of peptidylarginine deiminases (PADs), the enzymes mediating citrullination, through BB-Cl-amidine treatment, prevents diabetes development in NOD mice. This prevention was associated with reduced levels of citrullination in the pancreas, decreased circulating autoantibody titers against citrullinated glucose-regulated protein 78, and reduced spontaneous neutrophil extracellular trap formation of bone marrow-derived neutrophils. Moreover, BB-Cl-amidine treatment induced a shift from Th1 to Th2 cytokines in the serum and an increase in the frequency of regulatory T cells in the blood and spleen. In the pancreas, BB-Cl-amidine treatment preserved insulin production and was associated with a less destructive immune infiltrate characterized by reduced frequencies of effector memory CD4+ T cells and a modest reduction in the frequency of interferon-γ-producing CD4+ and CD8+ T cells. Our results point to a role of citrullination in the pathogenesis of autoimmune diabetes, with PAD inhibition leading to disease prevention through modulation of immune pathways. These findings provide insight in the potential of PAD inhibition for treating autoimmune diseases like type 1 diabetes.

Intestinal Delivery of Proinsulin and IL-10 via Lactococcus lactis Combined With Low-Dose Anti-CD3 Restores Tolerance Outside the Window of Acute Type 1 Diabetes Diagnosis.

A combination treatment (CT) of proinsulin and IL-10 orally delivered via genetically modified Lactococcus lactis bacteria combined with low-dose anti-CD3 (aCD3) therapy successfully restores glucose homeostasis in newly diagnosed non-obese diabetic (NOD) mice. Tolerance is accompanied by the accumulation of Foxp3+ regulatory T cells (Tregs) in the pancreas. To test the potential of this therapy outside the window of acute diabetes diagnosis, we substituted autoimmune diabetic mice, with disease duration varying between 4 and 53 days, with syngeneic islets at the time of therapy initiation. Untreated islet recipients consistently showed disease recurrence after 8.2 ± 0.7 days, while 32% of aCD3-treated and 48% of CT-treated mice remained normoglycemic until 6 weeks after therapy initiation (P < 0.001 vs. untreated controls for both treatments, P < 0.05 CT vs. aCD3 therapy). However, mice that were diabetic for more than 2 weeks before treatment initiation were less efficient at maintaining normoglycemia than those treated within 2 weeks of diabetes diagnosis, particularly in the aCD3-treated group. The complete elimination of endogenous beta cell mass with alloxan at the time of diabetes diagnosis pointed toward the significance of continuous feeding of the islet antigen proinsulin at the time of aCD3 therapy for treatment success. The CT providing proinsulin protected 69% of mice, compared to 33% when an irrelevant antigen (ovalbumin) was combined with aCD3 therapy, or to 27% with aCD3 therapy alone. Sustained tolerance was accompanied with a reduction of IGRP+CD8+ autoreactive T cells and an increase in insulin-reactive (InsB12-20 or InsB13-2) Foxp3+CD4+ Tregs, with a specific accumulation of Foxp3+ Tregs around the insulin-containing islet grafts after CT with proinsulin. The combination of proinsulin and IL-10 via oral Lactococcus lactis with low-dose aCD3 therapy can restore tolerance to beta cells in autoimmune diabetic mice, also when therapy is started outside the window of acute diabetes diagnosis, providing persistence of insulin-containing islets or prolonged beta cell function.

miR-409-3p is reduced in plasma and islet immune infiltrates of NOD diabetic mice and is differentially expressed in people with type 1 diabetes.

AIMS/HYPOTHESIS: MicroRNAs (miRNAs) are a novel class of potential biomarkers emerging in many diseases, including type 1 diabetes. Here, we aim to analyse a panel of circulating miRNAs in non-obese diabetic (NOD) mice and individuals with type 1 diabetes. METHODS: We adopted standardised methodologies for extracting miRNAs from small sample volumes to evaluate a profiling panel of mature miRNAs in paired plasma and laser-captured microdissected immune-infiltrated islets of recently diabetic and normoglycaemic NOD mice. Moreover, we validated the findings during disease progression and remission after anti-CD3 therapy in NOD mice, as well as in individuals with type 1 diabetes. RESULTS: Plasma levels of five miRNAs were downregulated in diabetic vs normoglycaemic mice. Of those, miR-409-3p was also downregulated in situ in the immune islet infiltrates of diabetic mice, suggesting an association with disease pathogenesis. Target-prediction tools linked miR-409-3p to immune- and metabolism-related signalling molecules. In situ miR-409-3p expression correlated with insulitis severity, and CD8+ central memory T cells were found to be enriched in miR-409-3p. Plasma miR-409-3p levels gradually decreased during diabetes development and improved with disease remission after anti-CD3 antibody therapy. Finally, plasma miR-409-3p levels were lower in people recently diagnosed with type 1 diabetes compared with a non-diabetic control group, and levels were inversely correlated with HbA1c levels. CONCLUSIONS/INTERPRETATION: We propose that miR-409-3p may represent a new circulating biomarker of islet inflammation and type 1 diabetes severity.

Screening for hypovitaminosis D: cost-effective or not?

Few topics have elicited more emotion than the issue of screening for vitamin D status and the discussion on the need for global supplementation with vitamin D metabolites. The importance of the problem is highlighted by the USPSTF posted draft research plan with the aim of making an update recommendations statement, possibly next year. Here, we discuss two different viewpoints on screening for vitamin D status: for and against. In the literature there are scientifically sound opinions supporting pro and cons positions. However, we believe that the best way to definitively elucidate this issue is the implementation of a randomized controlled trial evaluating clinical outcomes or harms in persons screened versus those not screened for vitamin D deficiency. The feasibility of such a trial is probably questionable owing to uncertainties still present concerning threshold for vitamin D sufficiency and end points (that is, for example, improved bone mineral density, reduced risk of falls and so on) to be reached.

Vitamin D-modulated dendritic cells delay lethal graft-versus-host disease through induction of regulatory T cells.

Graft-versus-host disease (GVHD) is the most lethal complication after allogeneic bone marrow transplantation (allo-BMT). Current approaches to prevent GVHD rely on donor lymphocyte/T cell depletion or general immunosuppression, leading to opportunistic infections and cancer relapse. Tolerogenic dendritic cells can induce regulatory T cells (Tregs) with the ability to suppress inflammation and prevent transplant rejection, making them an attractive cellular therapy to control GVHD. Active vitamin D (1α,25-dihydroxyvitamin D3; 1α,25(OH)2D3) promotes the generation of tolerogenic dendritic cells (1,25D3-DCs). This study aimed to determine the ability of ex vivo generated 1,25D3-DCs to trigger the expansion of Tregs that are able to control lethal xenogeneic GVHD in humanized NOD/LtSz-PrkdcscidIL2rγtm1Wjl (NSG) mice. We demonstrate that 1,25D3-DCs express lower levels of HLA-DR and costimulatory molecules, such as CD80 and CD86, and produce higher levels of IL-10 and TNF-α and lower amounts of IL-12, compared to vehicle-treated DCs. Moreover, these cells express increased levels of various co-inhibitory molecules such as PD-L1 and ILT-3 and the glycoprotein CD52 that is known to suppress T cell activation. Consequently, 1,25D3-DCs are poor stimulators of alloantigen-primed T cells, but foster the generation of antigen-specific suppressive Tregs. When adoptively transferred in humanized NSG mice, these 1,25D3-DC-induced Tregs delayed GVHD caused by the co-transferred autologous human peripheral blood mononuclear cells (PBMCs). These results indicate that 1,25D3-DC-induced Tregs can inhibit xenogeneic GVHD and maintain their immunomodulatory function under conditions of inflammation.

Vitamin D controls the capacity of human dendritic cells to induce functional regulatory T cells by regulation of glucose metabolism.

Tolerogenic dendritic cells (tolDCs) instruct regulatory T cells (Tregs) to dampen autoimmunity. Active vitamin D3 (1α,25-dihydroxyvitamin D3; 1α,25(OH)2D3) imprints human monocyte-derived DCs with tolerogenic properties by reprogramming their glucose metabolism. Here we identify the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) as a critical checkpoint and direct transcriptional target of 1α,25(OH)2D3 in determining the tolDC profile. Using tracer metabolomics, we show that PFKFB4 activity is essential for glucose metabolism, especially for glucose oxidation, which is elevated upon 1α,25(OH)2D3 exposure. Pharmacological inhibition of PFKFB4 reversed the 1α,25(OH)2D3-mediated shift in metabolism, DC profile and function, as determined by expression of inhibitory surface markers and secretion of regulatory cytokines and factors. Moreover, PFKFB4 inhibition in 1α,25(OH)2D3-treated DCs blocked their hallmark capacity to induce suppressive Tregs. This work demonstrates that alterations in the bioenergetic metabolism of immune cells are central to the immunomodulatory effects induced by 1α,25(OH)2D3.

Inflammation-Induced Citrullinated Glucose-Regulated Protein 78 Elicits Immune Responses in Human Type 1 Diabetes.

The ß-cell has become recognized as a central player in the pathogenesis of type 1 diabetes with the generation of neoantigens as potential triggers for breaking immune tolerance. We report that posttranslationally modified glucose-regulated protein 78 (GRP78) is a novel autoantigen in human type 1 diabetes. When human islets were exposed to inflammatory stress induced by interleukin-1ß, tumor necrosis factor-α, and interferon-γ, arginine residue R510 within GRP78 was converted into citrulline, as evidenced by liquid chromatography-tandem mass spectrometry. This conversion, known as citrullination, led to the generation of neoepitopes, which effectively could be presented by HLA-DRB1*04:01 molecules. With the use of HLA-DRB1*04:01 tetramers and ELISA techniques, we demonstrate enhanced antigenicity of citrullinated GRP78 with significantly increased CD4+ T-cell responses and autoantibody titers in patients with type 1 diabetes compared with healthy control subjects. Of note, patients with type 1 diabetes had a predominantly higher percentage of central memory cells and a lower percentage of effector memory cells directed against citrullinated GRP78 compared with the native epitope. These results strongly suggest that citrullination of ß-cell proteins, exemplified here by the citrullination of GRP78, contributes to loss of self-tolerance toward ß-cells in human type 1 diabetes, indicating that ß-cells actively participate in their own demise.

Inhibition of histone deacetylase 6 (HDAC6) protects against vincristine-induced peripheral neuropathies and inhibits tumor growth.

As cancer is becoming more and more a chronic disease, a large proportion of patients is confronted with devastating side effects of certain anti-cancer drugs. The most common neurological complications are painful peripheral neuropathies. Chemotherapeutics that interfere with microtubules, including plant-derived vinca-alkaloids such as vincristine, can cause these chemotherapy-induced peripheral neuropathies (CIPN). Available treatments focus on symptom alleviation and pain reduction rather than prevention of the neuropathy. The aim of this study was to investigate the potential of specific histone deacetylase 6 (HDAC6) inhibitors as a preventive therapy for CIPN using multiple rodent models for vincristine-induced peripheral neuropathies (VIPN). HDAC6 inhibition increased the levels of acetylated α-tubulin in tissues of rodents undergoing vincristine-based chemotherapy, which correlates to a reduced severity of the neurological symptoms, both at the electrophysiological and the behavioral level. Mechanistically, disturbances in axonal transport of mitochondria is considered as an important contributing factor in the pathophysiology of VIPN. As vincristine interferes with the polymerization of microtubules, we investigated whether disturbances in axonal transport could contribute to VIPN. We observed that increasing α-tubulin acetylation through HDAC6 inhibition restores vincristine-induced defects of axonal transport in cultured dorsal root ganglion neurons. Finally, we assured that HDAC6-inhibition offers neuroprotection without interfering with the anti-cancer efficacy of vincristine using a mouse model for acute lymphoblastic leukemia. Taken together, our results emphasize the therapeutic potential of HDAC6 inhibitors with beneficial effects both on vincristine-induced neurotoxicity, as well as on tumor proliferation.

MIF inhibition interferes with the inflammatory and T cell-stimulatory capacity of NOD macrophages and delays autoimmune diabetes onset.

Macrophages contribute in the initiation and progression of insulitis during type 1 diabetes (T1D). However, the mechanisms governing their recruitment into the islets as well as the manner of retention and activation are incompletely understood. Here, we investigated a role for macrophage migration inhibitory factor (MIF) and its transmembrane receptor, CD74, in the progression of T1D. Our data indicated elevated MIF concentrations especially in long-standing T1D patients and mice. Additionally, NOD mice featured increased MIF gene expression and CD74+ leukocyte frequencies in the pancreas. We identified F4/80+ macrophages as the main immune cells in the pancreas expressing CD74 and showed that MIF antagonism of NOD macrophages prevented their activation-induced cytokine production. The physiological importance was highlighted by the fact that inhibition of MIF delayed the onset of autoimmune diabetes in two different diabetogenic T cell transfer models. Mechanistically, macrophages pre-conditioned with the MIF inhibitor featured a refractory capacity to trigger T cell activation by keeping them in a naïve state. This study underlines a possible role for MIF/CD74 signaling pathways in promoting macrophage-mediated inflammation in T1D. As therapies directed at the MIF/CD74 pathway are in clinical development, new opportunities may be proposed for arresting T1D progression.

Reversal of Diabetes in NOD Mice by Clinical-Grade Proinsulin and IL-10-Secreting Lactococcus lactis in Combination With Low-Dose Anti-CD3 Depends on the Induction of Foxp3-Positive T Cells.

The introduction of ß-cell autoantigens via the gut through Lactococcus lactis (L. lactis) has been demonstrated to be a promising approach for diabetes reversal in NOD mice. Here we show that a combination therapy of low-dose anti-CD3 with a clinical-grade self-containing L. lactis, appropriate for human application, secreting human proinsulin and interleukin-10, cured 66% of mice with new-onset diabetes, which is comparable to therapy results with plasmid-driven L. lactis Initial blood glucose concentrations (<350 mg/dL) and insulin autoantibody positivity were predictors of the stable reversal of hyperglycemia, and decline in insulin autoantibody positivity was an immune biomarker of therapeutic outcome. The assessment of the immune changes induced by the L. lactis-based therapy revealed elevated frequencies of CD4+Foxp3+ T cells in the pancreas-draining lymph nodes, pancreas, and peripheral blood of all treated mice, independent of metabolic outcome. Neutralization of cytotoxic T-lymphocyte antigen 4 and transforming growth factor-ß partially abrogated the suppressive function of therapy-induced regulatory T cells (Tregs). Ablation or functional impairment of Foxp3+ Tregs in vivo at the start or stop of therapy impaired immune tolerance, highlighting the dependence of the therapy-induced tolerance in mice with new-onset diabetes on the presence and functionality of CD4+Foxp3+ T cells. Biomarkers identified in this study can potentially be used in the future to tailor the L. lactis-based combination therapy for individual patients.

Lactococcus lactis As a Versatile Vehicle for Tolerogenic Immunotherapy.

Genetically modified Lactococcus lactis bacteria have been engineered as a tool to deliver bioactive proteins to mucosal tissues as a means to exert both local and systemic effects. They have an excellent safety profile, the result of years of human consumption in the food industry, as well as a lack of toxicity and immunogenicity. Also, containment strategies have been developed to promote further application as clinical protein-based therapeutics. Here, we review technological advancements made to enhanced the potential of L. lactis as live biofactories and discuss some examples of tolerogenic immunotherapies mediated by mucosal drug delivery via L. lactis. Additionally, we highlight their use to induce mucosal tolerance by targeted autoantigen delivery to the intestine as an approach to reverse autoimmune type 1 diabetes.

Klugiella xanthotipulae gen. nov., sp. nov., a novel member of the family Microbacteriaceae.

An actinobacterium, designated strain 44C3(T), was isolated in Michigan, USA, from the hindgut of the larvae of Tipula abdominalis, an aquatic crane fly, and was subjected to a polyphasic taxonomic investigation. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain represented a separate clade within the family Microbacteriaceae. It showed highest 16S rRNA gene sequence similarity with Cryobacterium psychrotolerans 0549(T) (96.5 %). Strain 44C3(T) had a novel B-type peptidoglycan. The peptidoglycan contained the diamino acid lysine, the peptide Gly-d-Glu was detected in the partial hydrolysate and alanine was the N terminus of the interpeptide bridge. No other amino acids found in other B-type peptidoglycans (including diaminobutyric acid, ornithine, homoserine and hydroxyglutamic acid) could be detected. The major menaquinones were MK-12 and MK-11, the major fatty acids were ai-C(15 : 0), ai-C(17 : 0) and i-C(16 : 0) and the DNA G+C content was 60.9 mol%. Analysis of the chemotaxonomic and phylogenetic data suggested that strain 44C3(T) represented a novel species of a new genus within the family Microbacteriaceae, for which the name Klugiella xanthotipulae gen. nov., sp. nov. is proposed. The type strain of Klugiella xanthotipulae is 44C3(T) (=DSM 18031(T) =ATCC BAA-1524(T)).

Microbial conversion of sugars from plant biomass to lactic acid or ethanol.

Concerns for our environment and unease with our dependence on foreign oil have renewed interest in converting plant biomass into fuels and 'green' chemicals. The volume of plant matter available makes lignocellulose conversion desirable, although no single isolated organism has been shown to depolymerize lignocellulose and efficiently metabolize the resulting sugars into a specific product. This work reviews selected chemicals and fuels that can be produced from microbial fermentation of plant-derived cell-wall sugars and directed engineering for improvement of microbial biocatalysts. Lactic acid and ethanol production are highlighted, with a focus on engineered Escherichia coli.

Isolation of polymer-degrading bacteria and characterization of the hindgut bacterial community from the detritus-feeding larvae of Tipula abdominalis (Diptera: Tipulidae).

The Tipula abdominalis larval hindgut microbial community presumably facilitates digestion of the lignocellulosic diet. The microbial community was investigated through characterization of bacterial isolates and analysis of 16S rRNA gene clone libraries. This initial study revealed novel bacteria and provides a framework for future studies of this symbiosis.