Studying Arthritis and Microbes - A Research Proposal

What follows is a research proposal that aims to address certain immunological questions with regards to how the immune system can cause autoimune arthritis, and how this disease process is shaped by the microbes that live in our gut. For a companion piece to this proposal that may provide a broader introduction to this issue and why it is important for both scientific resarch and human health, please see the article Human Interactions with Microbes

Background and Introduction

The average adult human digestive tract contains roughly 100 trillion microbial cells, 99.1% of which are estimated to be bacterial (Qin et al., 2010). Despite these high levels of commensal microbes, healthy individuals do not experience a chronic state of intestinal inflammation in part due to a number of bacterial immunomodulatory effector functions. Many enteric species have been shown to downregulate signaling of the proinflammatory transcription factor NF-κB in intestinal cells by means of a variety of unique mechanisms, ultimately serving to attenuate host inflammatory pathways (O'Hara and Shanahan, 2006). Other bacteria are able to evade host defense mechanisms by mimicking certain molecular moieties of the gut (O'Hara and Shanahan, 2006). The colonization of commensal flora is also associated with protection of the host against enteropathogenic species, in part due to the ability of these commensal microbes to outcompete pathogenic strains for resources including nutrients and space (Guarner and Malagelada, 2003).

Perhaps unsurprisingly, commensal microbes have been implicated in autoimmune diseases of the digestive tract including Crohn’s disease and ulcerative colitis, largely due to the aberrant functioning of enteric tolerance mechanisms resulting in a state of chronic inflammation (O'Hara and Shanahan, 2006). More recently and unexpectedly, however, germ-free mice lacking any enteric bacteria have been shown to be resistant to the progression of a number of autoimmune diseases not classically associated with the gut, including autoimmune arthritis and experimental autoimmune encephalomyelitis (Wu, et al., 2010; Lee, et al., 2011).

The mechanism by which commensal microbes are thought to mediate this onset of distal autoimmune disease stems from the ability of certain enteric species to induce the differentiation of a class of proinflammatory CD4+ T lymphocytes known as Th17 cells. Classically T helper cells were identified as being of either Th1 or Th2 phenotype, with autoimmune disease resulting from a perturbation of the Th1/Th2 balance. More recently, however, IL-17 secreting T helper cells (Th17 cells) were identified and were implicated in a number of autoimmune diseases originally attributed to Th1/Th2 imbalance. IL-17 knockout mice are resistant to the onset of a range of autoimmune disorders including collagen induced arthritis, experimental myocarditis, and allergic airway hypersensitivity, clearly demonstrating a critical role for this molecule, and thus for these cells, in disease progression (Steinman, 2007).

Th17 cells characteristically express the transcription factor RORγt and secrete the proinflammatory cytokines IL-17A, IL-17F, IL-21, and IL-22 (Steinman, 2007). This inflammatory cytokine profile offers insight into why Th1 and Th2 cells must tightly regulate Th17 differentiation, and suggests an explanation as to why Th17 activity may be integral to the progression of inflammatory autoimmune diseases. IL-21, for example, has been implicated in the development and maintenance of B cell germinal centers which are necessary for the progression of many autoimmune diseases (Zotos, et al., 2010). Germ-free mice lacking normal Th17 cell levels present with correspondingly reduced splenic B cell germinal center populations, and consequently experience a significant reduction in autoimmune arthritic progression, illustrating a mechanism by which Th17 cells mediate autoimmunity in vivo (Wu, et al., 2010).

While Th17 cells are normally enriched in the small intestine lamina propria (SI-LP) of mice, they are found systemically at low levels, and up to 50% of splenic Th17 cells express the gut homing integrin α4β7 (Lochner, et al., 2011). This attests to the intestinal origin of these cells, likely explaining why imbalances in gut flora can have systemic Th17-mediated consequences. Indeed, germ-free mice have a significantly reduced Th17 cell levels both in the spleen and the SI-LP, and at least one study has demonstrated a time-course of Th17 activity such that Th17 levels are initially elevated only in the SI-LP, followed one week later by an increase in splenic Th17 cells, followed one week later by a significant increase in the presentation of arthritic symptoms (Wu, et al., 2010; Lochner, et al., 2011).

Commensal microbes are critical for this development of a normal systemic population of Th17 cells, especially in the SI-LP, however Th17 differentiation does not depend upon the presentation of microbial antigens to naïve CD4+ lymphocytes, suggesting that there must be an alternative mechanism of microbe-mediated differentiation (Lochner, et al., 2011). Specific bacteria, particularly those of the poorly-characterized family of Clostridia-related gram positive microbes known as segmented filamentous bacteria (SFB), have been strongly associated with Th17 cell induction in the SI-LP in vivo (Ivanov, et al. 2009). Monocolonization of mice with SFB restored a relatively normal Th17 population to previously germ-free mice which had been lacking a normal Th17 complement, resulting in a corresponding increase in B cell germinal centers and a subsequent onset of arthritis not present in mice colonized with unrelated bacteria (Wu, et al., 2010). SFB are known to be able to penetrate the mucosal lining of the gut moreso than most bacteria, associating directly and strongly with receptors on the surface of intestinal epithelial cells (Umesaki and Setoyama, 2000). As Th17 differentiation is not antigen-dependent, it has been proposed that this direct binding plays a critical role in this differentiation, explaining why SFB are a superior inducer of Th17-mediated autoimmunity.

While bacterially induced Th17 cells undoubtedly serve important functions in healthy individuals, their proinflammatory potential underscores the need to tightly regulate them so as to prevent chronic inflammatory diseases. In addition to being downregulated by the major Th1 and Th2 cytokines IFNγ and IL-4, Th17 differentiation is dependent on a number of other cytokines including TGF-β, IL-6, and IL-23 (Veldhoen, et al., 2006). Recent studies suggest that the differentiation of regulatory T cells (Tregs) and Th17 cells are reciprocal processes, as TGF-β expression is critical to the development of both of these cell types (Ziegler and Buckner, 2009). Both of these cells are in part characterized by their expression of specific transcription factors – RORγt for Th17 cells and FoxP3 for Treg cells – and it has been proposed that the relative expression of these transcription factors in naïve CD4+ T cells is what ultimately determines cell fate (Ziegler and Buckner, 2009). For example, it has been shown in vitro that retinoic acid treatment drives increased FoxP3 expression and leads to a corresponding increase in Treg cells (Xiao, et al., 2008). Treatment with retinoic acid in vivo leads to the α4β7-mediated gut homing of Tregs and the suppression of Th17-mediated experimental autoimmune encephalomyelitis (Benson et al., 2007; Xiao, et al., 2008). Interestingly, retinoic acid is a major byproduct of vitamin A metabolism by gut bacteria, suggesting another mechanism by which commensal flora may regulate autoimmune disease progression in vivo.

Recently, a subset of CD4+ T cells expressing both RORγt and FoxP3 has been identified, and while these cells were initially interpreted as Th17/Treg intermediates, they have since been shown to have unique and physiologically relevant properties. Foxp3+ RORγt+ T cells characteristically express IL-10, an anti-inflammatory cytokine, and have been shown to function as a subset of regulatory T cells in vitro (Lochner, et al., 2008). Furthermore, these cells were shown to remain in a constant equilibrium with Th17 cells in vivo during both infected and healthy states, and an expansion of the Foxp3+ RORγt+ cell population is suppressive of autoimmune diabetes (Lochner, et al., 2008; Tartar, et al., 2010). Unlike Th17 cells, normal Foxp3+ RORγt+ T cell differentiation is dependent on the presentation of cognate gut microbial antigens, suggesting yet another critical function for enteric bacteria in the prevention of autoimmune disease (Lochner, et al., 2011). Given this past work, it seems very likely that Foxp3+ RORγt+ T cells will be suppressive of a number of autoimmune disorders and may thus serve as a relevant avenue of study with regard to disease prevention and treatment.

Autoimmune arthritis is a valuable model disease to study in murine populations, as its progression can be readily monitored and its symptomology often closely mimics that of a common human form of rheumatoid arthritis (RA). RA is a highly debilitating autoimmune polyarthritis affecting 0.5-1% of people, leading to joint destruction and a consequent decline in quality of life for these affected individuals (Smolen and Steiner, 2003). The prevalence and severity of this disease makes it a crucial target for novel clinical interventions and related research, with the ultimate goal of identifying new ways to both treat the disorder and to prevent its initial development. In order to enhance the clinical value of these studies, it is important to utilize an experimental model of autoimmune arthritis which is decidedly similar to human RA, as is the case for the K/BxN mice used herein. Like RA but unlike many other mouse models of arthritis such as collagen-induced arthritis (CIA), K/BxN mice spontaneously develop arthritis beginning at an age of 30±5 days (Monach et al. 2007). This arthritis stems from a systemic immune response to the endogenous protein glucose-6-phosphate isomerase, leading to a disease progression similar to that of RA, characterized by spontaneous autoantibody production, leukocyte invasion of joints, bone/cartilage erosion, and polyclonal B cell activation (Monach et al. 2007). These mice do not, however, express a biomarker typical of RA known as rheumatoid factor, demonstrating that while this model system is a valuable research tool, there are some differences between K/BxN autoimmune arthritis in mice and RA in humans.

K/BxN mice have previously been studied in the context of Th17-mediated autoimmunity. Germ-free K/BxN mice develop a significantly attenuated arthritis relative to age/sex-matched specific pathogen-free K/BxN mice. These germ-free mice were additionally shown to be deficient in Th17 cells, and neutralization of IL-17 was found to eliminate disease progression in specific pathogen-free mice, as would be expected for a Th17-mediated autoimmune disease (Wu, et al. 2010). This Th17-dependent disease progression likely stems, in part, from a reduction in B cell germinal centers in Th17-deficient mice due to a resultant decrease in IL-21 secretion. This theory is supported by studies demonstrating that germ-free/Th17-deficient K/BxN mice present with fewer and smaller splenic germinal centers, and that K/BxN mice deficient in IL-21 experience no appreciable arthritic progression (Wu, et al., 2010; Jang, et al,. 2009).

Despite the extensive study of Th17 cells in the context of K/BxN arthritis, to the best of our knowledge no groups have expressly studied Foxp3+ RORγt+ T cell activity in these mice in the context of disease progression. This, coupled with the previous demonstration of the autoimmune diabetes-suppressing potential of these cells and the established relationship between Th17 cells and these T cell intermediates, suggests that Foxp3+ RORγt+ cells will likely be suppressive of other Th17-mediated autoimmune disorders including K/BxN autoimmune arthritis.

Taken together, the abovementioned studies clearly demonstrate the instrumental role of commensal bacteria in the induction of both Th17 and Foxp3+ RORγt+ T cells in vivo, with perturbations in the balance of these two cell lines serving as a potential factor in the onset of autoimmune diseases including autoimmune arthritis. As such, they exhort the need to assess whether arthritic progression can be arrested by modulating levels of these cells in at-risk patient populations, thereby significantly improving quality of life for millions of affected individuals. These studies will serve as just such an assessment by altering the Th17/ Foxp3+ RORγt+ T cell balance in K/BxN mice in vivo, thereby providing direct insight into the clinical relevance and medicinal value of these cell populations and opening the door to a multitude of future research opportunities.

Experimental Approach

  • Establishment of germ-free and specific pathogen-free NOD-K/BxN mice
  • Confirmation of the dependence of NOD-K/BxN arthritic onset on the presence of commensal microbiota and Th17 activity
  • Expansion of Treg cells in vivo using all-trans retinoic acid to assess the effects of this expansion on systemic T cell populations and arthritic progression, and to assess the levels of FoxP3+ RORγt+ cells among expanded Tregs
  • Assessment of the effects of FoxP3+ RORγt+ T cells adoptively transferred from NOD mice on arthritic progression and T cell populations in specific pathogen free K/BxN mice
  • Study of the feasibility of expanding FoxP3+ RORγt+ T cells in vivo in K/BxN mice using a previously described tolerogenic Ig-GAD1 chimera

Establishment of germ-free and specific pathogen-free NOD-K/BxN mice

In order to characterize the regulatory role of Foxp3+ RORγt+ T cells on arthritic progression in K/BxN mice as a function of both bacterial load and Th17 cell levels, it will be necessary to establish both germ-free (GF) and specific pathogen-free (SPF) colonies of K/BxN mice. Production of K/BxN mice requires the establishment of breeding colonies of both KRN TCR transgenic mice and NOD mice. Typically, KRN TCR transgenic mice are of a B6 background, however some of the experiments in this study will necessitate K/BxN mice of a syngenic NOD background. As K/BxN arthritis has been shown to arise from the recognition of glucose-6-phosphate isomerase by the KRN TCR in the context of the NOD-derived Ag7 MHC Class II molecule, it will be necessary to obtain NOD mice which MHC class II-deficient (Monach, et al. 2007). To this end, NOD mice lacking the class II transactivator protein (CIIA) which have been shown to express extremely low levels of MHC class II (0.8% of splenocytes vs. 31% in WT mice) will be obtained through collaboration with the Mora research group at Yale University in New Haven, CT (Mora, et al., 1999). KRN TCR transgenic mice of the B6 background will be bred with NOD.CIIA:KO mice, and CIIA deficient offspring expressing the KRN TCR (as assessed by PCR; for primers see Mora, et al., 1999 and Monach, et al., 2007) will be backcrossed onto NOD mice for 10 generations in order to obtain NOD.KRN.CIIA:KO mice.

In order to obtain arthritic NOD-K/BxN mice, NOD.KRN.CIIA:KO males will be bred with WT-NOD females, and offspring will be genotyped via PCR using the primers detailed by Mora et al. and Monach et al. in order to establish which offspring express the CIIA protein and the transgenic TCR necessary for spontaneous arthritic onset (hereafter referred to as NOD-K/BxN mice) and which do not (hereafter referred to as BxN mice) (Mora, et al., 1999; Monach, et al., 2007). SPF mice will be maintained in the specific pathogen-free facilities of the University of Connecticut in sterile laminar flow cages on 12 hour day/night cycles with free access to food and water.

GF NOD-K/BxN mice will be produced by establishing GF colonies of both NOD.KRN.CIIA:KO and WT-NOD mice which will be bred and genotyped as in SPF conditions. Initial GF mice will be produced by cesarean rederivation and will be raised by a GF foster mother in GF facilities at Taconic Farms (Germantown, NY) as previously described (Wu, et al. 2010). These mice will be regularly screened for commensal microbiota by culturing fecal samples aerobically or anaerobically for one week at 37oC and examining samples for microbial growth of any kind (Inzunza et al. 2005). Mice will be shipped to the University of Connecticut in GF containers.

Additionally, previously described NOD.FoxP3:GFP reporter mice will be obtained through collaboration with researchers at the University of Missouri of Columbia (Tartar, et al. 2010). If these mice are unavailable, a new colony will be established using the methods of Tartar, et al. by breeding C57BL6.FoxP3:GFP knock-in mice into NOD mice and backcrossing for 10 generations. These mice will be maintained in SPF conditions. Studies making use of animals will be executed according to the guidelines established by the University of Connecticut Institutional Animal Care and Use Committee.

Confirmation of the Dependence of NOD-K/BxN Arthritic Onset on the Presence of Commensal Microbiota and Th17 Activity

Before further studies are conducted using GF and SPF NOD-K/BxN mice, it is important to confirm that the findings of Wu, et al. hold true in our NOD-K/BxN mice by demonstrating that arthritis occurs in SPF mice and is attenuated in GF mice. Additionally we must confirm that GF mice have reduced levels of Th17 cells, and that arthritic progression in SPF mice is dependent on Th17 cell activity.

First, arthritic progression in both GF and SPF NOD-K/BxN mice will be assessed by clinical index, ankle thickening, and anti-GPI autoantibody titer as measured by ELISA over time (Wu, et al., 2010). This experiment is expected to confirm that arthritis is reduced in GF mice, particularly early in disease progression. Differences in Th17 and FoxP3+RORγt+ T cells between these two populations will be determined using flow cytometry as previously described, in order to confirm the effect of commensal gut flora on T cell differentiation (Lochner, et al., 2011). Briefly, cells from the SI-LP and spleens of age/sex-matched GF and SPF mice will be homogenized, stimulated in vitro for 5 hours with PMA + ionomyicin, fixed, permeabilized, labeled with appropriately conjugated mAbs (purchased from Invitrogen), and analyzed in the University of Connecticut flow cytometry facilities. Th17 cells will be defined as CD4+ TCRβ+ RORγt+ IL17+ IL10- Foxp3- cells, and FoxP3+RORγt+ cells will be defined as CD4+ TCRβ+ RORγt+ FoxP3+ IL10+ IL17- cells (Lochner, et al., 2011). GF mice are expected to have a significantly reduced Th17 cell population, and are thus likely to have a proportionately reduced FoxP3+RORγt+ cell population in order to maintain the equilibrium of these two cell types (Lochner, et al. 2008).

In order to demonstrate the Th17 cell-dependence of arthritis in NOD-K/BxN mice, NOD-K/BxN mice will be sacrificed and the spleens and lymph nodes will be used to prepare single cell suspensions which can be labeled with fluorescently tagged antibodies and sorted using fluorescence activated cell sorting (FACS) in order to isolate naïve CD4+ T cells (Veldhoen, et al., 2006). These cells will then be incubated with TGFβ1, IL-6, and IL-23 (purchased from Sigma) in appropriate culture media in order to promote Th17 differentiation of these cells in vitro (Veldhoen, et al., 2006). Th17 phenotype should be confirmed using flow cytometry, and remaining CD4+ TCRβ+ IL17+ IFNγ- cells should be isolated by FACS. These cells should then be adoptively transferred into age/sex-matched GF or SPF K/BxN mice by injecting 3 x 105 cells or sterile saline buffer into the tail veins of these mice (Heuer et al., 2005). Non-control mice will additionally be injected every 3 days with anti-IL-17 or an isotype-matched control Ig in order to assess the effects of the neutralization of IL-17 function (Wu, et al., 2010). Arthritic progression will be monitored in these mice over time. Additionally, mice will be sacrificed every 7 days and the splenic and SI-LP CD4+ T cell populations will be quantified by flow cytometry.

Potential Pitfalls and Alternative Approaches

This set of experiments is expected to confirm that arthritic progression in NOD-K/BxN mice is dependent on Th17 activity, as previously shown. Additionally, the adoptive transfer of Th17 cells and IL-17 neutralization will provide valuable insight into the role of Th17 induction on both disease progression and T cell differentiation in the presence and absence of commensal microbes. Conducting these experiments should be feasible, although expanding Th17 cells in vitro may require significant optimization. If difficulty is encountered in these experiments, other techniques will be employed such as the stimulating of Th17 populations in vivo using LPS, a technique currently being studied at the University of Connecticut Health Center in Farmington, CT (McAleer, et al., 2010).

Expansion of Treg cells in vivo using all-trans retinoic acid to assess the effects of this expansion on systemic T cell populations and arthritic progression, and to assess the levels of FoxP3+ RORγt+ cells among expanded Tregs

It has previously been shown that administration of all-trans retinoic acid (ATRA) to mice is protective against the inflammation associated with ulcerative colitis and experimental autoimmune encephalomyelitis (Xiao, et al., 2008; Bai, et al., 2009). ATRA induces Treg differentiation in vitro and Treg gut homing in vivo, and studies have indicated that it abrogates inflammation by altering the Th17/Treg balance (Xiao, et al., 2008; Benson, et al., 2007; Bai, et al., 2009). To the best of our knowledge, no studies to date have assessed the levels of FoxP3+RORγt+ T cells among ATRA-induced gut homing Treg cells.

In order to elucidate this area of limited understanding, SPF NOD-K/BxN mice will be treated daily with an intraperitoneal injection of either ATRA (purchased from Sigma) dissolved in DMSO or of media control (Bai, et al., 2009). Treatment will begin at time points of either 1,3, or 5 weeks of age in order to establish the time-dependence of any observed effects. Arthritic progression will be monitored in differently treated mice. Mice will be sacrificed at an age of 7 weeks and the levels of Th17 and FoxP3+RORγt+ T cells in the SI-LP and spleen will be characterized by flow cytometry as before. If these experiments yield increased levels of FoxP3+RORγt+ Tregs, GF NOD-K/BxN mice will undergo the same treatment regimen in order to assess the necessity of commensal microbes for normal Treg induction following ATRA treatment.

Potential Pitfalls and Alternative Approaches

This set of experiments will serve to improve current understanding of the role of ATRA in specific Treg induction and subsequent regulation of Th17-mediated autoimmunity. It cannot be guaranteed that the ATRA-induced Treg cell population will be enriched with FoxP3+RORγt+ cells. Previous studies of the induction of Tregs in NOD mice demonstrated that these Tregs were suppressive of autoimmune diabetes largely due to an increased level of FoxP3+RORγt+ Tregs (Tartar, et al., 2010). These studies made use of an Ig-chimera which promoted Treg expansion and pancreatic localization; a localization which was necessary to confer a suppression of autoimmune diabetes (Tartar, et al., 2010). These results suggest that ATRA will be a similarly effective inducer of FoxP3+RORγt+ Tregs, and that these induced Tregs will localize with gut-induced Th17 cells resulting in protection against the onset of autoimmune arthritis in K/BxN mice. Even if ATRA does not induce significant levels of FoxP3+RORγt+ T cells, the resultant data will improve current understanding of Treg dynamics and of time-dependent suppression of Th17-mediated autoimmune arthritis by Tregs in NOD-K/BxN mice.

Assessment of the effects of FoxP3+ RORγt+ T cells adoptively transferred from NOD mice on arthritic progression and T cell populations in specific pathogen free NOD-K/BxN mice

The previously mentioned experiments conducted by Tartar et al. have successfully demonstrated a technique capable of expanding FoxP3+RORγt+ Tregs in vivo in NOD mice. This technique relies upon the use of an Ig-GAD1 chimera which induces immune tolerance to the GAD1 protein via a Treg-mediated mechanism, ultimately protecting against autoimmune diabetes (Gregg, et al., 2004). Further studies demonstrated that induced Tregs included a significant fraction of cells expressing both FoxP3 and RORγt, and that these live cells could be purified using FACS when expressed in a NOD.FoxP3:GFP reporter mice due to their characteristic GFP expression levels (Tartar, et al., 2010).

In order to assess whether these cells can be adoptively transferred to NOD-K/BxN mice and what effect they may have upon arthritic progression, it will first be necessary to obtain aliquots of the Ig-GAD1 chimera. This will be achieved either in collaboration with the Tartar research group or by replicating their previously described methods in order to produce this protein (Gregg, et al., 2004). NOD.FoxP3:GFP will then receive i.p. injections of the protein at 4,5, and 6 weeks of age, and will be sacrificed at the end of 6 weeks (Tartar, et al., 2010). In pilot studies, cells of the spleen, pancreas, and pancreatic lymph nodes will be fixed and permeabilized in order to test for RORγt expression using specific fluorescently tagged antibodies, and will additionally be stained for CD4 and membrane-bound TGFβ (Tartar, et al., 2010). These cells will be assessed by flow cytometry so as to establish whether or not there is a direct correlation between intracellular levels of GFP and the FoxP3+RORγt+ Treg phenotype; in the study conducted by Tartar et al., these cells consistently produced intermediate level of GFP which allowed for them to be purified from other CD4+ T cells on this basis. After proper gating protocols have been established so as to allow for the identification of relatively pure (>95%) FoxP3+RORγt+ Tregs, live CD4+ cells will be sorted by FACS based on GFP expression levels in order to purify these regulatory cells of interest.

Saline solution or purified FoxP3+RORγt+ Tregs derived from NOD mice will be intravenously injected into the tail vein of GF or SPF NOD-K/BxN mice at either 1,3, or 5 weeks of age, and arthritic progression in these mice will be monitored over time. Some mice will be sacrificed after 7 weeks and the levels of Th17 cells and FoxP3+RORγt+ Tregs in the spleen, pancreas, and SI-LP will be assessed by flow cytometry. This analysis will include an assessment of GFP expression so as to establish the fates of the NOD.FoxP3:GFP purified FoxP3+RORγt+ Tregs. If a suppression of autoimmune arthritis is observed in SPF conditions, segmented filamentous bacteria (SFB)-monoassociated mice (established as by Wu, et al. 2010) will undergo the same adoptive transfer regimen so as to demonstrate that these monoassociated bacteria can restore arthritic phenotype to GF mice, and that this arthritis can be suppressed by FoxP3+RORγt+ T cells.

Potential Pitfalls and Alternative Approaches

These experiments will likely demonstrate the successful adoptive transfer of FoxP3+RORγt+ Tregs into NOD-K/BxN mice, providing direct insight into the disease suppressing role of these cells on Th17 differentiation and arthritic progression. Of particular interest will be the relative efficacy of adoptive transfer/disease suppression in GF mice when compared to SPF and SFB-monoassociated mice. It is possible, however, that endogenous Treg cells may impede adoptively transferred Treg activity. An alternative approach will be to use antibody injection to deplete endogenous CD4+CD25+ Tregs as previously described prior to adoptive transfer of FoxP3+RORγt+ Tregs (Kang, et al., 2007). This should allow for an enhanced determination of the role of these cells in the context of Th17-mediated autoimmune arthritis progression.

Study of the feasibility of expanding FoxP3+ RORγt+ T cells in vivo in NOD-K/BxN mice using a previously described tolerogenic Ig-GAD1 chimera

To the best of our knowledge, the Ig-GAD1 chimera has only been utilized as a means of expanding the endogenous levels of FoxP3+RORγt+ Tregs in NOD mice. As our K/BxN mice are of a syngenic NOD-background, it is highly likely that this expansion will occur in vivo in these mice following Ig-GAD1 exposure. In order to test this assumption, GF or SPF NOD-K/BxN mice will be treated with Ig-GAD1 or saline solution by i.p. injection starting at 1,3, or 5 weeks of age, and arthritic progression will be monitored over time. Mice will be sacrificed at an age of 7 weeks and the levels of FoxP3+RORγt+ Tregs in the pancreas, spleen, and SI-LP will be assessed by flow cytometry. Additionally, levels of mRNA transcripts characteristic of Th17 or FoxP3+RORγt+ Tregs (IL-17, IL-10, CCR6, CCL20, FoxP3, RORγt) among splenic and SI-LP CD4+ T cells will be assessed by real-time PCR as previously described (Lochner, et al., 2010). This will allow for an alternative means of confirmation of the different levels of these cells in Ig-GAD1-exposed mice relative to controls.

As in the previous set of experiments, if a reduction in arthritic progression occurs in SPF mice following FoxP3+RORγt+ Treg expansion and significantly different T cell populations are established in SPF mice as compared to GF mice, these experiments will be replicated in SFB-monoassociated mice. Doing so will allow for a definitive demonstration of the ability to modulate SFB-induced Th17-mediated arthritic progression in vivo via the induction of FoxP3+RORγt+ Tregs.

Potential Pitfalls and Alternative Approaches

These experiments are expected to demonstrate that FoxP3+RORγt+ Tregs can be generated in our NOD-K/BxN arthritic mice, and that these cells are able to suppress to progression of autoimmune arthritis by downregulating Th17 cell activity. Assuming the Ig-GAD1 procedures were successfully optimized in NOD.FoxP3:GFP mice, these experiments should be relatively straightforward. It may be valuable to additionally confirm the regulatory function of these FoxP3+RORγt+ Tregs by depleting the CD4+CD25+ Treg cells before Ig-GAD1 induction using anti-CD25 treatment as previously described and assessing the onset of arthritis in these mice relative to those in which Tregs were not depleted (Kang, et al., 2007). This will provide further confirmation of the relevance of FoxP3+RORγt+ Tregs as a subset of other Treg cells in the context of disease progression. Ultimately, this set of experiments is expected to confirm the disease-relevance of FoxP3+RORγt+ Tregs and to thus open the door to research into myriad potential treatments for autoimmune inflammatory disorders. If the results of these experiments are not as expected, they will still provide valuable insight into the intricacies of Th17 and Treg function in the context of autoimmune disease development and progression, greatly improving the current body of knowledge regarding this subject and laying the foundations for future research.


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