Inverse-Vaccines for Rheumatoid Arthritis Re-establish Metabolic and Immunological Homeostasis in Joint Tissues.

Rheumatoid arthritis (RA) causes immunological and metabolic imbalances in tissue, exacerbating inflammation in affected joints. Changes in immunological and metabolic tissue homeostasis at different stages of RA are not well understood. Herein, the changes in the immunological and metabolic profiles in different stages in collagen induced arthritis (CIA), namely, early, intermediate, and late stage is examined. Moreover, the efficacy of the inverse-vaccine, paKG(PFK15+bc2) microparticle, to restore tissue homeostasis at different stages is also investigated. Immunological analyses of inverse-vaccine-treated group revealed a significant decrease in the activation of pro-inflammatory immune cells and remarkable increase in regulatory T-cell populations in the intermediate and late stages compared to no treatment. Also, glycolysis in the spleen is normalized in the late stages of CIA in inverse-vaccine-treated mice, which is similar to no-disease tissues. Metabolomics analyses revealed that metabolites UDP-glucuronic acid and L-Glutathione oxidized are significantly altered between treatment groups, and thus might provide new druggable targets for RA treatment. Flux metabolic modeling identified amino acid and carnitine pathways as the central pathways affected in arthritic tissue with CIA progression. Overall, this study shows that the inverse-vaccines initiate early re-establishment of homeostasis, which persists through the disease span.

Engineered Tumor-Immune Microenvironment On A Chip to Study T Cell-Macrophage Interaction in Breast Cancer Progression.

Evolving knowledge about the tumor-immune microenvironment (TIME) is driving innovation in designing novel therapies against hard-to-treat breast cancer. Targeting the immune components of TIME has emerged as a promising approach for cancer therapy. While recent immunotherapies aim at restoring antitumor immunity, counteracting tumor escape remains challenging. Hence there is a pressing need to better understand the complex tumor-immune crosstalk within TIME. Considering this imperative, this study aims at investigating the crosstalk between the two abundant immune cell populations within the breast TIME-macrophages and T cells, in driving tumor progression using an organotypic 3D in vitro tumor-on-a-chip (TOC) model. The TOC features distinct yet interconnected organotypic tumor and stromal entities. This triculture platform mimics the complex TIME, embedding the two immune populations in a suitable 3D matrix. Analysis of invasion, morphometric measurements, and flow cytometry results underscores the substantial contribution of macrophages to tumor progression, while the presence of T cells is associated with a deceleration in the migratory behavior of both cancer cells and macrophages. Furthermore, cytokine analyses reveal significant upregulation of leptin and RANTES (regulated on activation, normal T Cell expressed and secreted) in triculture. Overall, this study highlights the complexity of TIME and the critical role of immune cells in cancer progression.

Employing Feature Selection Algorithms to Determine the Immune State of Mice Model of Rheumatoid Arthritis.

The immune response is a dynamic process by which the body determines whether an antigen is self or nonself. The state of this dynamic process is defined by the relative balance and population of inflammatory and regulatory actors which comprise this decision making process. The goal of immunotherapy as applied to, e.g. Rheumatoid Arthritis (RA), then, is to bias the immune state in favor of the regulatory actors - thereby shutting down autoimmune pathways in the response. While there are several known approaches to immunotherapy, the effectiveness of the therapy will depend on how this intervention alters the evolution of this state. Unfortunately, this process is determined not only by the dynamics of the process, but the state of the system at the time of intervention - a state which is difficult if not impossible to determine prior to application of the therapy. To identify such states we consider a mouse model of RA (Collagen-Induced Arthritis (CIA)) immunotherapy; collect high dimensional data on T cell markers and populations of mice after treatment with a recently developed immunotherapy for CIA; and use feature selection algorithms in order to select a lower dimensional subset of this data which can be used to predict both the full set of T cell markers and populations, along with the efficacy of immunotherapy treatment.

Rescue of dendritic cells from glycolysis inhibition improves cancer immunotherapy in mice.

Inhibition of glycolysis in immune cells and cancer cells diminishes their activity, and thus combining immunotherapies with glycolytic inhibitors is challenging. Herein, a strategy is presented where glycolysis is inhibited in cancer cells using PFK15 (inhibitor of PFKFB3, rate-limiting step in glycolysis), while simultaneously glycolysis and function is rescued in DCs by delivery of fructose-1,6-biphosphate (F16BP, one-step downstream of PFKFB3). To demonstrate the feasibility of this strategy, vaccine formulations are generated using calcium-phosphate chemistry, that incorporate F16BP, poly(IC) as adjuvant, and phosphorylated-TRP2 peptide antigen and tested in challenging and established YUMM1.1 tumours in immunocompetent female mice. Furthermore, to test the versatility of this strategy, adoptive DC therapy is developed with formulations that incorporate F16BP, poly(IC) as adjuvant and mRNA derived from B16F10 cells as antigens in established B16F10 tumours in immunocompetent female mice. F16BP vaccine formulations rescue DCs in vitro and in vivo, significantly improve the survival of mice, and generate cytotoxic T cell (Tc) responses by elevating Tc1 and Tc17 cells within the tumour. Overall, these results demonstrate that rescuing glycolysis of DCs using metabolite-based formulations can be utilized to generate immunotherapy even in the presence of glycolytic inhibitor.

Exponentially decreasing exposure of antigen generates anti-inflammatory T-cell responses.

Rheumatoid Arthritis (RA) is a chronic debilitating disease characterized by auto-immune reaction towards self-antigen such as collagen type II. In this study, we investigated the impact of exponentially decreasing levels of antigen exposure on pro-inflammatory T cell responses in the collagen-induced arthritis (CIA) mouse model. Using a controlled delivery experimental approach, we manipulated the collagen type II (CII) antigen concentration presented to the immune system. We observed that exponentially decreasing levels of antigen generated reduced pro-inflammatory T cell responses in secondary lymphoid organs in mice suffering from RA. Specifically, untreated mice exhibited robust pro-inflammatory T cell activation and increased paw inflammation, whereas, mice exposed to exponentially decreasing concentrations of CII demonstrated significantly reduced pro-inflammatory T cell responses, exhibited lower levels of paw inflammation, and decreased arthritis scores in right rear paw. The data also demonstrate that the decreasing antigen levels promoted the induction of regulatory T cells (Tregs), which play a crucial role in maintaining immune tolerance and suppressing excessive inflammatory responses. Our findings highlight the importance of antigen concentration in modulating pro-inflammatory T cell responses in the CIA model. These results provide valuable insights into the potential therapeutic strategies that target antigen presentation to regulate immune responses and mitigate inflammation in rheumatoid arthritis and other autoimmune diseases. Further investigations are warranted to elucidate the specific mechanisms underlying the antigen concentration-dependent modulation of T cell responses and to explore the translational potential of this approach for the development of novel therapeutic interventions in autoimmune disorders.

Succinate in the tumor microenvironment affects tumor growth and modulates tumor associated macrophages.

Succinate is an important metabolite that modulates metabolism of immune cells and cancer cells in the tumor microenvironment (TME). Herein, we report that polyethylene succinate (PES) microparticles (MPs) biomaterial mediated controlled delivery of succinate in the TME modulates macrophage responses. Administering PES MPs locally with or without a BRAF inhibitor systemically in an immune-defective aging mice with clinically relevant BRAFV600E mutated YUMM1.1 melanoma decreased tumor volume three-fold. PES MPs in the TME also led to maintenance of M1 macrophages with up-regulation of TSLP and type 1 interferon pathway. Impressively, this led to generation of pro-inflammatory adaptive immune responses in the form of increased T helper type 1 and T helper type 17 cells in the TME. Overall, our findings from this challenging tumor model suggest that immunometabolism-modifying PES MP strategies provide an approach for developing robust cancer immunotherapies.

Succinate based polymers drive immunometabolism in dendritic cells to generate cancer immunotherapy.

Boosting the metabolism of immune cells while restricting cancer cell metabolism is challenging. Herein, we report that using biomaterials for the controlled delivery of succinate metabolite to phagocytic immune cells activates them and modulates their metabolism in the presence of metabolic inhibitors. In young immunocompetent mice, polymeric microparticles, with succinate incorporated in the backbone, induced strong pro-inflammatory anti-melanoma responses. Administration of poly(ethylene succinate) (PES MP)-based vaccines and glutaminase inhibitor to young immunocompetent mice with aggressive and large, established B16F10 melanoma tumors increased their survival three-fold, a result of increased cytotoxic T cells expressing RORγT (Tc17). Mechanistically, PES MPs directly modulate glutamine and glutamate metabolism, upregulate succinate receptor SUCNR1, activate antigen presenting cells through and HIF-1alpha, TNFa and TSLP-signaling pathways, and are dependent on alpha-ketoglutarate dehydrogenase for their activity, which demonstrates correlation of succinate delivery and these pathways. Overall, our findings suggest that immunometabolism-modifying PES MP strategies provide an approach for developing robust cancer immunotherapies.

Characterization of 2 Novel Phosphodiesterase 2 Inhibitors Hcyb1 and PF-05180999 on Depression- and Anxiety-Like Behavior.

BACKGROUND: Phosphodiesterase 2A (PDE2A) represents a novel target for new therapies addressing psychiatric disorders. To date, the development of PDE2A inhibitors suitable for human clinical evaluation has been hampered by the poor brain accessibility and metabolic stability of the available compounds. METHODS: Corticosterone (CORT)-induced neuronal cell lesion and restraint stress mouse model were used to measure the neuroprotective effect in cells and antidepressant-like behavior in mice. RESULTS: The cell-based assay showed that both Hcyb1 and PF were potent in protecting cells against stress hormone CORT insults by stimulating cAMP and cGMP signaling in hippocampal cells (HT-22). Administration of both compounds before treatment of CORT to cells increased cAMP/cGMP, VASP phosphorylation at Ser239 and Ser157, cAMP response element binding protein phosphorylation at Ser133, and brain derived neurotrophic factor BDNF expression. Further in vivo study showed that both Hcyb1 and PF displayed -antidepressant- and anxiolytic-like effects against restraint stress as indicated by reduced immobility time in the forced swimming and tail suspension tasks as well as increased open arm entries and time spent in open arms and holes visit in elevated plus maze and hole-board tests, respectively. The biochemical study confirmed that these antidepressant- and anxiolytic-like effects of Hcyb1 and PF were related to cAMP and cGMP signaling in the hippocampus. CONCLUSIONS: The results extend the previous studies and validate that PDE2A is a tractable target for drug development in the treatment of emotional disorders such as depression and anxiety.

Alpha-ketoglutaric acid based polymeric particles for cutaneous wound healing.

Metabolites are not only involved in energy pathways but can also act as signaling molecules. Herein, we demonstrate that polyesters of alpha-ketoglutararte (paKG) can be generated by reacting aKG with aliphatic diols of different lengths, which release aKG in a sustained manner. paKG polymer-based microparticles generated via emulsion-evaporation technique lead to faster keratinocyte wound closures in a scratch assay test. Moreover, paKG microparticles also led to faster wound healing responses in an excisional wound model in live mice. Overall, this study shows that paKG MPs that release aKG in a sustained manner can be used to develop regenerative therapeutic responses.

Vaccines prevent reinduction of rheumatoid arthritis symptoms in collagen-induced arthritis mouse model.

Metabolic reprogramming of immune cells modulates their function and reduces the severity of autoimmune diseases. However, the long-term effects of the metabolically reprogrammed cells, specifically in the case of immune flare-ups, need to be examined. Herein, a re-induction rheumatoid arthritis (RA) mouse model was developed by injecting T-cells from RA mice into drug-treated mice to recapitulate the effects of T-cell-mediated inflammation and mimic immune flare-ups. Immune metabolic modulator paKG(PFK15 + bc2) microparticles (MPs) were shown to reduce clinical symptoms of RA in collagen-induced arthritis (CIA) mice. Upon re-induction, a significant delay in the reappearance of clinical symptoms in the paKG(PFK15 + bc2) microparticle treatment group was observed as compared to equal or higher doses of the clinically utilized U.S. Food and Drug Administration (FDA)-approved drug, Methotrexate (MTX). Furthermore, paKG(PFK15 + bc2) microparticle-treated mice were able to lower activated dendritic cells (DCs) and inflammatory T helper cell 1 (TH1) and increased activated, proliferating regulatory T-cells (Tregs) more effectively than MTX. The paKG(PFK15 + bc2) microparticles also led to a significant reduction in paw inflammation in mice as compared to MTX treatment. This study can pave the way for the development of flare-up mouse models and antigen-specific drug treatments.

Immune response differences in degradable and non-degradable alloy implants.

Alloy based implants have made a great impact in the clinic and in preclinical research. Immune responses are one of the major causes of failure of these implants in the clinic. Although the immune responses toward non-degradable alloy implants are well documented, there is a poor understanding of the immune responses against degradable alloy implants. Recently, there have been several reports suggesting that degradable implants may develop substantial immune responses. This phenomenon needs to be further studied in detail to make the case for the degradable implants to be utilized in clinics. Herein, we review these new recent reports suggesting the role of innate and potentially adaptive immune cells in inducing immune responses against degradable implants. First, we discussed immune responses to allergen components of non-degradable implants to give a better overview on differences in the immune response between non-degradable and degradable implants. Furthermore, we also provide potential areas of research that can be undertaken that may shed light on the local and global immune responses that are generated in response to degradable implants.

Biomaterial mediated simultaneous delivery of spermine and alpha ketoglutarate modulate metabolism and innate immune cell phenotype in sepsis mouse models.

Although different metabolic pathways have been associated with distinct macrophage phenotypes, the field of utilizing metabolites to modulate macrophage phenotype is in a nascent stage. In this report, we developed microparticles based on polymerization of alpha-ketoglutarate (a Krebs cycle metabolite), with or without encapsulation of spermine (a polyamine metabolite), to modulate cell phenotype that are critical for resolution of inflammation. Poly (alpha-ketoglutarate) microparticles encapsulated and released spermine (spermine (encap)paKG MPs) in vitro, which was accelerated in an acidic environment. When delivered to bone marrow-derived-macrophages, spermine (encap)paKG MPs induced a complex phenotypic profile outside of the typical M1/M2 paradigm, with distinct effects in the presence or absence of the pro-inflammatory stimulus lipopolysaccharide. Of particular interest was the increase in expression of CD163, which has been linked to anti-inflammatory responses in sepsis. Therefore, we systemically administered spermine (encap)paKG MPs to two different murine models of sepsis using acute or chronic injection of LPS. Macrophages and neutrophils in the liver and spleen of animals treated with spermine (encap)paKG MPs increased expression of CD163, concomitant with normalizing of glycolysis and oxidative phosphorylation, in both models. Overall, these results show that spermine (encap)paKG MPs modulate macrophage phenotype in vitro and in vivo, with potential applications in inflammation-associated diseases.

Short term, low dose alpha-ketoglutarate based polymeric nanoparticles with methotrexate reverse rheumatoid arthritis symptoms in mice and modulate T helper cell responses.

Activated effector T cells induce pro-inflammatory responses in rheumatoid arthritis (RA) which then lead to inflammation of the joints. In this report, we demonstrate that polymeric nanoparticles with alpha keto-glutarate (aKG) in their polymer backbone (termed as paKG NPs) modulate T cell responses in vitro and in vivo. Impressively, a low dose of only three administrations of methotrexate, a clinically and chronically administered drug for RA, in conjunction with two doses of paKG NPs, reversed arthritis symptoms in collagen-induced arthritis (CIA) mice. This was further followed by significant decreases in pro-inflammatory antigen-specific T helper type 17 (TH17) responses and a significant increase in anti-inflammatory regulatory T cell (TREG) responses when CIA treated splenic cells were isolated and re-exposed to the CIA self-antigen. Overall, this study supports the concurrent and short term, low dose of paKG NPs and methotrexate for the reversal of RA symptoms.

In silico identification and synthesis of a multi-drug loaded MOF for treating tuberculosis.

Conventional drug delivery systems have been applied to a myriad of active ingredients but may be difficult to tailor for a given drug. Herein, we put forth a new strategy, which designs and selects the drug delivery material by considering the properties of encapsulated drugs (even multiple drugs, simultaneously). Specifically, through an in-silico screening process of 5109 MOFs using grand canonical Monte Carlo simulations, a customized MOF (referred as BIO-MOF-100) was selected and experimentally verified to be biologically stable, and capable of loading 3 anti-Tuberculosis drugs Rifampicin+Isoniazid+Pyrazinamide at 10% + 28% + 23% wt/wt (total > 50% by weight). Notably, the customized BIO-MOF-100 delivery system cleared naturally Pyrazinamide-resistant Bacillus Calmette-Guérin, reduced growth of virulent Erdman infection in macaque macrophages 10-100-fold compared to soluble drugs in vitro and was also significantly reduced Erdman growth in mice. These data suggest that the methodology of identifying-synthesizing materials can be used to generate solutions for challenging applications such as simultaneous delivery of multiple, small hydrophilic and hydrophobic molecules in the same molecular framework.

Drug delivery for metabolism targeted cancer immunotherapy.

Drug delivery vehicles have made a great impact on cancer immunotherapies in clinics and pre-clinical research. Notably, the science of delivery of cancer vaccines and immunotherapeutics, modulating immune cell functions has inspired development of several successful companies and clinical products. Interestingly, these drug delivery modalities not only modulate the function of immune cells (often quantified at the mRNA and protein levels), but also modulate the metabolism of these cells. Specifically, cancer immunotherapy often leads to activation of different immune cells such as dendritic cells, macrophages and T cells, which is driven by energy metabolism of these cells. Recently, there has been a great excitement about interventions that can directly modulate the energy metabolism of these immune cells and thus affect their function and in turn lead to a robust cancer immune response. Here we review few strategies that have been tested in clinic and pre-clinical research for generating effective metabolism-associated cancer therapies and immunotherapies.

Oral drug delivery for immunoengineering.

The systemic pharmacotherapeutic efficacy of immunomodulatory drugs is heavily influenced by its route of administration. A few common routes for the systemic delivery of immunotherapeutics are intravenous, intraperitoneal, and intramuscular injections. However, the development of novel biomaterials, in adjunct to current progress in immunoengineering, is providing an exciting area of interest for oral drug delivery for systemic targeting. Oral immunotherapeutic delivery is a highly preferred route of administration due to its ease of administration, higher patient compliance, and increased ability to generate specialized immune responses. However, the harsh environment and slow systemic absorption, due to various biological barriers, reduces the immunotherapeutic bioavailability, and in turn prevents widespread use of oral delivery. Nonetheless, cutting edge biomaterials are being synthesized to combat these biological barriers within the gastrointestinal (GI) tract for the enhancement of drug bioavailability and targeting the immune system. For example, advancements in biomaterials and synthesized drug agents have provided distinctive methods to promote localized drug absorption for the modulation of local or systemic immune responses. Additionally, novel breakthroughs in the immunoengineering field show promise in the development of vaccine delivery systems for disease prevention as well as combating autoimmune diseases, inflammatory diseases, and cancer. This review will discuss current progress made within the field of biomaterials and drug delivery systems to enhance oral immunotherapeutic availability, and how these new delivery platforms can be utilized to deliver immunotherapeutics for resolution of immune-related diseases.

GRAS-microparticle microarrays identify dendritic cell tolerogenic marker-inducing formulations.

Microarrays, miniaturized platforms used for high-content studies, provide potential advantages over traditional in vitro investigation in terms of time, cost, and parallel analyses. Recently, microarrays have been leveraged to investigate immune cell biology by providing a platform with which to systematically investigate the effects of various agents on a wide variety of cellular processes, including those giving rise to immune regulation for application toward curtailing autoimmunity. A specific embodiment incorporates dendritic cells cultured on microarrays containing biodegradable microparticles. Such an approach allows immune cell and microparticle co-localization and release of compounds on small, isolated populations of cells, enabling a quick, convenient method to quantify a variety of cellular responses in parallel. In this study, the microparticle microarray platform was utilized to investigate a small library of sixteen generally regarded as safe (GRAS) compounds (ascorbic acid, aspirin, capsaicin, celastrol, curcumin, epigallocatechin-3-gallate, ergosterol, hemin, hydrocortisone, indomethacin, menadione, naproxen, resveratrol, retinoic acid, α-tocopherol, vitamin D3) for their ability to induce suppressive phenotypes in murine dendritic cells. Two complementary tolerogenic index ranking systems were proposed to summarize dendritic cell responses and suggested several lead compounds (celastrol, ergosterol, vitamin D3) and two secondary compounds (hemin, capsaicin), which warrant further investigation for applications toward suppression and tolerance.

Inhibition of glycolysis in the presence of antigen generates suppressive antigen-specific responses and restrains rheumatoid arthritis in mice.

Dendritic cells (DCs) rely on glycolysis for their energy needs to induce pro-inflammatory antigen-specific immune responses. Therefore, inhibiting DC glycolysis, while presenting the self-antigen, may prevent pro-inflammatory antigen-specific immune responses. Previously we demonstrated that microparticles with alpha-ketoglutarate (aKG) in the polymer backbone (paKG MPs) were able to generate anti-inflammatory DCs by sustained delivery of the aKG metabolite, and by modulating energy metabolism of DCs. Herein, we demonstrate that paKG MPs-based delivery of a glycolytic inhibitor, PFK15, using paKG MPs induces anti-inflammatory DCs (CD86LoMHCII+) by down-regulating glycolysis, CD86, tnf and IL-6 genes, while upregulating oxidative phosphorylation (OXPHOS) and mitochondrial genes. Furthermore, paKG MPs delivering PFK15 and a self-antigen, collagen type II (bc2), in vivo, in a collagen-induced autoimmune arthritis (CIA) mouse model, normalized paw inflammation and arthritis score, by generating antigen-specific immune responses. Specifically, these formulations were able to reduce activation of DCs in draining lymph nodes and impressively generated proliferating bc2-specific anti-inflammatory regulatory T cells in joint-associated popliteal lymph nodes. These data strongly suggest that sustained glycolytic inhibition of DCs in the presence of an antigen can induce antigen-specific immunosuppressive responses, therefore, generating a technology that can be applicable for treating autoimmune diseases.

Instruction of Immunometabolism by Adipose Tissue: Implications for Cancer Progression.

Disruption of metabolic homeostasis at the organismal level can cause metabolic syndrome associated with obesity. The role of adipose tissue in cancer has been investigated over the last several decades with many studies implicating obesity as a risk factor for the development of cancer. Adipose tissue contains a diverse array of immune cell populations that promote metabolic homeostasis through a tightly controlled balance of pro- and anti-inflammatory signals. During obesity, pro-inflammatory cell types infiltrate and expand within the adipose tissue, exacerbating metabolic dysfunction. Some studies have now shown that the intracellular metabolism of immune cells is also deregulated by the lipid-rich environment in obesity. What is not fully understood, is how this may influence cancer progression, metastasis, and anti-tumor immunity. This review seeks to highlight our current understanding of the effect of adipose tissue on immune cell function and discuss how recent results offer new insight into the role that adipose tissue plays in cancer progression and anti-tumor immunity.