Innate immunomodulation with MIS416: mechanism of action in experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an immune-mediated disease in which self-reacting T lymphocytes enter the central nervous system (CNS) and direct the damage of the myelin sheaths, which protect nerve axons. While there is no cure for MS currently, specific disease-modifying therapies are available that target the relapsing-remitting form of MS. However, these therapies are not effective in progressive forms of MS, which affect ~50% of MS patients in New Zealand, and thus there is an urgent need for novel treatments to be developed to treat these patients. MIS416 is a microparticle, which targets phagocytic cells by activating cytosolic receptors NOD2 and Toll-like receptor 9, and has recently completed a phase 2a trial in the treatment of progressive MS with promising results. The aim of this thesis was to elucidate the mechanism(s) by which MIS416 modified autoimmune disease using an animal model of MS, experimental autoimmune encephalomyelitis (EAE). We determined that weekly intravenous doses of MIS416 were effective at reducing disease in a chronic model of MS, EAE, and we used this dosing regimen to further understand mechanisms involved in MIS416-induced EAE protection. It was found that MIS416 treatment induced a number of peripheral immune changes which had the potential to alter a T cell-mediated autoimmune disease, including increased levels of serum NO and IFN-γ, increased numbers of Tregs and macrophages as well as increased expression of PD-L1, an anti-proliferative ligand. Additionally, we also determined that MIS416 treatment significantly reduced T cell proliferation in vitro and in vivo. To investigate which of these peripheral changes were involved in MIS416-mediated protection from EAE, we assessed each change individually in our disease model. We found that the central immune-modulating factor responsible for the protective effects of MIS416 treatment was IFN-γ (White, Webster, O'Sullivan, Stone, & La Flamme, 2014) as IFN-γ-/- mice treated with MIS416 were not protected from EAE. This protective role of MIS416-induced IFN-γ was likely to have been mediated through downstream effects given that in the absence of IFN-γ many of these changes were not seen. In particular, the macrophage population did not expand in MIS416-treated IFN-γ-/- mice, and the expression of the immunoregulatory ligand, PD-L1, was not enhanced suggesting that macrophage expressed PD-L1 may be one of the ways in which MIS416-induced IFN-γ altered EAE. While NO was found to contribute to the MIS416-mediated suppression of T cell proliferation ex vivo and IFN-γ-/- mice did not have elevated levels of NO, the in vivo inhibition of iNOS by aminoguanidine did not ablate MIS416 protection suggesting that these pathways were not central to disease protection. Additionally, MIS416 treatment was shown to increase the number and function of Tregs, and in the absence of IFN-γ this expansion was reduced. Altogether, this research indicated that IFN-γ was key to MIS416-mediated protection possibly through an expansion of immunoregulatory macrophage populations, enhanced PD-L1 expression, and enhanced Treg numbers and function. In addition to these peripheral immune effects, we found that MIS416 treatment also altered cellular trafficking to the CNS. MIS416-treated EAE mice had reduced CNS infiltration as measured by both flow cytometry and histology compared to untreated EAE mice, and MIS416 treatment also reduced the EAE-induced permeability of the blood brain barrier. Furthermore, our study determined that MIS416-induced trafficking of immune cells to the CNS in the absence of EAE, and this trafficking also occurred in an IFN-γ-dependent manner. Given the suppressive nature of cells found within the periphery of MIS416-treated mice, the cells which trafficked to the CNS in response to MIS416 treatment may have had beneficial roles in EAE by suppressing T cell responses from within the CNS. In summary, the work outlined in this thesis revealed that MIS416 treatment induced an immunoregulatory state that was capable of suppressing T cell proliferation and reducing EAE disease. Moreover, this research highlighted that in the right context, a pro-inflammatory cytokine such as IFN-γ may be protective in autoimmune disease. Finally, when these findings are applied more widely, they indicate that the immune modulations induced by MIS416 may have potential in other diseases mediated by immune dysregulation in addition to MS

The parasite cytokine mimic <i>Hp</i>-TGM potently replicates the regulatory effects of TGF-β on murine CD4+ T cells

Transforming growth factor‐beta (TGF‐β) family proteins mediate many vital biological functions in growth, development and regulation of the immune system. TGF‐β itself controls immune homeostasis and inflammation, including conversion of naïve CD4+ T cells into Foxp3+ regulatory T cells (Tregs) in the presence of IL‐2 and T cell receptor ligands. The helminth parasite Heligmosomoides polygyrus exploits this pathway through a structurally novel TGF‐β mimic (Hp‐TGM), which binds to mammalian TGF‐β receptors and induces Tregs. Here, we performed detailed comparisons of Hp‐TGM with mammalian TGF‐β. Compared to TGF‐β, Hp‐TGM induced greater numbers of Foxp3+ Tregs (iTregs), with more intense Foxp3 expression. Both ligands upregulated Treg functional markers CD73, CD103 and PD‐L1, but Hp‐TGM induced significantly higher CD39 expression than did TGF‐β. Interestingly, in contrast to canonical TGF‐β signalling through Smad2/3, Hp‐TGM stimulation was slower and more sustained. Gene expression profiles induced by TGF‐β and Hp‐TGM were remarkably similar, and both types of iTregs suppressed T cell responses in vitro and EAE‐driven inflammation in vivo. In vitro, both types of iTregs were equally stable under inflammatory conditions, but Hp‐TGM‐induced iTregs were more stable in vivo during DSS‐induced colitis, with greater retention of Foxp3 expression and lower conversion to a ROR‐γt+ phenotype. Altogether, results from this study suggest that the parasite cytokine mimic, Hp‐TGM, may deliver a qualitatively different signal to CD4+ T cells with downstream consequences for the long‐term stability of iTregs. These data highlight the potential of Hp‐TGM as a new modulator of T cell responses in vitro and in vivo

The helminth parasite heligmosomoides polygyrus attenuates EAE in an IL-4Rα-dependent manner

Helminth parasites are effective in biasing Th2 immunity and inducing regulatory pathways that minimize excessive inflammation within their hosts, thus allowing chronic infection to occur whilst also suppressing bystander atopic or autoimmune diseases. Multiple sclerosis (MS) is a severe autoimmune disease characterized by inflammatory lesions within the central nervous system; there are very limited therapeutic options for the progressive forms of the disease and none are curative. Here, we used the experimental autoimmune encephalomyelitis (EAE) model to examine if the intestinal helminth Heligmosomoides polygyrus and its excretory/secretory products (HES) are able to suppress inflammatory disease. Mice infected with H. polygyrus at the time of immunization with the peptide used to induce EAE (myelin-oligodendrocyte glycoprotein, pMOG), showed a delay in the onset and peak severity of EAE disease, however, treatment with HES only showed a marginal delay in disease onset. Mice that received H. polygyrus 4 weeks prior to EAE induction were also not significantly protected. H. polygyrus secretes a known TGF-β mimic (Hp-TGM) and simultaneous H. polygyrus infection with pMOG immunization led to a significant expansion of Tregs; however, administering the recombinant Hp-TGM to EAE mice failed to replicate the EAE protection seen during infection, indicating that this may not be central to the disease protecting mechanism. Mice infected with H. polygyrus also showed a systemic Th2 biasing, and restimulating splenocytes with pMOG showed release of pMOG-specific IL-4 as well as suppression of inflammatory IL-17A. Notably, a Th2-skewed response was found only in mice infected with H. polygyrus at the time of EAE induction and not those with a chronic infection. Furthermore, H. polygyrus failed to protect against disease in IL-4Rα−/− mice. Together these results indicate that the EAE disease protective mechanism of H. polygyrus is likely to be predominantly Th2 deviation, and further highlights Th2-biasing as a future therapeutic strategy for MS

TGF-β mimic proteins form an extended gene family in the murine parasite Heligmosomoides polygyrus

We recently reported the discovery of a new parasite-derived protein that functionally mimics the immunosuppressive cytokine transforming growth factor (TGF)-β. The Heligmosomoides polygyrus TGF-β Mimic (Hp-TGM) shares no homology to any TGF-β family member, however it binds the mammalian TGF-β receptor and induces expression of Foxp3, the canonical transcription factor of both mouse and human regulatory T cells. Hp-TGM consists of five atypical Complement Control Protein (CCP, Pfam 00084) domains, each lacking certain conserved residues and 12–15 amino acids longer than the 60–70 amino acids consensus domain, but with a recognizable 3-cysteine, tryptophan, cysteine motif. We now report on the identification of a family of nine related Hp-TGM homologues represented in the secreted proteome and transcriptome of H. polygyrus. Recombinant proteins from five of the nine new TGM members were tested for TGF-β activity, but only two were functionally active in an MFB-F11 reporter assay, and by the induction of T cell Foxp3 expression. Sequence comparisons reveal that proteins with functional activity are similar or identical to Hp-TGM across the first three CCP domains, but more variable in domains 4 and 5. Inactive proteins diverged in all domains, or lacked some domains entirely. Testing truncated versions of Hp-TGM confirmed that domains 1–3 are essential for full activity in vitro, while domains 4 and 5 are not required. Further studies will elucidate whether these latter domains fulfill other functions in promoting host immune regulation during infection and if the more divergent family members play other roles in immunomodulation

Witness Response at Acute Onset of Stroke: A Qualitative Theory-Guided Study

Background: Delay in calling emergency medical services following stroke limits access to early treatment that can reduce disability. Emergency medical services contact is mostly initiated by stroke witnesses (often relatives), rather than stroke patients. This study explored appraisal and behavioural factors that are potentially important in influencing witness behaviour in response to stroke. Methods and Findings: Semi-structured interviews with 26 stroke witnesses were transcribed and theory-guided content analysed was undertaken based on the Common Sense Self-Regulation Model (appraisal processes) and Theory Domains Framework (behavioural determinants). Response behaviours were often influenced by heuristics-guided appraisal (i.e. mental rules of thumb). Some witnesses described their responses to the situation as &lsquo;automatic' and &lsquo;instinctive', rather than products of deliberation. Potential behavioural influences included: environmental context and resources (e.g. time of day), social influence (e.g. prompts from patients) and beliefs about consequences (e.g. 999 accesses rapid help). Findings are based on retrospective accounts and need further verification in prospective studies. Conclusions: Witnesses play a key role in patient access to emergency medical services. Factors that potentially influence witnesses' responses to stroke were identified and could inform behavioural interventions and future research. Interventions might benefit from linking automatic/instinctive threat perceptions with deliberate appraisal of stroke symptoms, prompting action to call emergency medical services

Induction of stable human FOXP3⁺ Tregs by a parasite-derived TGF-β mimic

Immune homeostasis in the intestine is tightly controlled by FOXP3 + regulatory T cells (Tregs), defects of which are linked to the development of chronic conditions, such as inflammatory bowel disease (IBD). As a mechanism of immune evasion, several species of intestinal parasites boost Treg activity. The parasite Heligmosomoides polygyrus is known to secrete a molecule (Hp-TGM) that mimics the ability of TGF-β to induce FOXP3 expression in CD4 + T cells. The study aimed to investigate whether Hp-TGM could induce human FOXP3 + Tregs as a potential therapeutic approach for inflammatory diseases. CD4 + T cells from healthy volunteers were expanded in the presence of Hp-TGM or TGF-β. Treg induction was measured by flow cytometric detection of FOXP3 and other Treg markers, such as CD25 and CTLA-4. Epigenetic changes were detected using ChIP-Seq and pyrosequencing of FOXP3. Treg phenotype stability was assessed following inflammatory cytokine challenge and Treg function was evaluated by cellular co-culture suppression assays and cytometric bead arrays for secreted cytokines. Hp-TGM efficiently induced FOXP3 expression (&gt; 60%), in addition to CD25 and CTLA-4, and caused epigenetic modification of the FOXP3 locus to a greater extent than TGF-β. Hp-TGM-induced Tregs had superior suppressive function compared with TGF-β-induced Tregs, and retained their phenotype following exposure to inflammatory cytokines. Furthermore, Hp-TGM induced a Treg-like phenotype in in vivo differentiated Th1 and Th17 cells, indicating its potential to re-program memory cells to enhance immune tolerance. These data indicate Hp-TGM has potential to be used to generate stable human FOXP3 + Tregs to treat IBD and other inflammatory diseases. </p

The impact of the UK 'Act FAST' stroke awareness campaign: Content analysis of patients, witness and primary care clinicians' perceptions

Background: The English mass media campaign &lsquo;Act FAST' aimed to raise stroke awareness and the need to call emergency services at the onset of suspected stroke. We examined the perceived impact and views of the campaign in target populations to identify potential ways to optimise mass-media interventions for stroke. Methods: Analysis of semi-structured interviews conducted as part of two qualitative studies, which examined factors influencing patient/witness response to acute stroke symptoms (n&thinsp;=&thinsp;19 stroke patients, n&thinsp;=&thinsp;26 stroke witnesses) and perceptions about raising stroke awareness in primary care (n&thinsp;=&thinsp;30 clinicians). Both studies included questions about the &lsquo;Act FAST' campaign. Interviews were content analysed to determine campaign awareness, perceived impact on decisions and response to stroke, and views of the campaign. Results: Most participants were aware of the Act FAST campaign. Some patients and witnesses reported that the campaign impacted upon their stroke recognition and response, but the majority reported no impact. Clinicians often perceived campaign success in raising stroke awareness, but few thought it would change response behaviours. Some patients and witnesses, and most primary care clinicians expressed positive views towards the campaign. Some more critical participant comments included perceptions of dramatic, irrelevant, and potentially confusing content, such as a prominent &lsquo;fire in the brain' analogy. Conclusions: Act FAST has had some perceived impact on stroke recognition and response in some stroke patients and witnesses, but the majority reported no campaign impact. Primary care clinicians were positive about the campaign, and believed it had impacted on stroke awareness and recognition but doubted impact on response behaviour. Potential avenues for optimising and complementing mass media campaigns such as &lsquo;Act FAST' were identified