When Little Things Make a Big Difference

Introduction

The immune system, which encompasses a dynamic network of organs, cells, and molecules, functions to maintain homeostasis as well as to respond to threats. Regulation of the immune system at the genetic, epigenetic and functional level is achieved by a multitude of cellular and acellular factors, collectively referred to as immune modulatory agents. Any natural or artificial disruption of this delicate balance creates ripple effects that have local and/or systemic manifestations which steer the outcome towards a protective or a pathological immune response. A clear and in-depth understanding of immune modulatory agents and their mechanisms of action is critical as it allows us to address or correct immune dysfunction, and such timely intervention can have far-reaching implications in human health and disease. The current thematic issue of Immunological Investigations focuses on both positive and negative modulation of the immune system by acellular particles or particulates that range in size from a few nanometers to a few micrometers, and are accordingly defined as nanoparticles (between 1 and 1000 nm) or microparticles (between 1 and 1000 µm).

While micro and nanoparticles are extremely diverse, certain common features make them unique immune modulatory agents. Firstly, their size range allows them to be taken up by antigen presenting cells (APCs) either by endocytosis (for smaller particles that are 20–200 nm), activating the cell-mediated branch of the immune system, or by phagocytosis and macro-pinocytosis (for larger particles that are 0.5–5 µm), which activate the humoral branch (Xiang et al. 2006). Secondly, relatively high permeability of these particles allows delivery of antigens and/or immune modulatory agents across biological barriers. Thirdly, particulates can protect the integrity of the associated molecules, preventing degradation until they are delivered to the immune cells. In addition to these common characteristics, each particle has unique properties depending on its origin, composition and contents, all of which contribute to its impact on the immune system.

The long and ever-growing list of immune modulatory microparticles and nanoparticles can be broadly classified into intrinsic and extrinsic particles depending on their origin. Intrinsic particles originate within the body and include vesicles secreted by cells, collectively termed as extracellular vesicles (EVs). Extrinsic particles on the other hand, are introduced into the body from the external environment, and may be naturally occurring particles that are accidentally introduced (e.g. Asian Sand Dust or ASD that is often inhaled), or synthetic/engineered particles (e.g. Virus-like particles or VLPs, PLGA nanoparticles, lipid-based nanoparticles), that are deliberately introduced to boost immune responses. This thematic issue of Immunological Investigations provides an eclectic sampling of immune modulatory micro and nanoparticles from all of the above categories, and features a rich assortment of articles from a variety of disciplines such as tumor immunology, biotechnology, inflammation, virology, transplantation, vaccines and immunotherapy.

Climate change has resulted in an increase in the incidence of dust storms, and this, together with the ability of dust to travel long distances translates into a growing number of people being exposed to dust. Although the size as well as chemical and mineral composition of dust vary depending on its origin, all dusts are capable of carrying contaminants such as pollutants, microbes, and microbial products, exacerbating the hazards associating with its inhalation. As a result, exposure to dust storms is often followed by an increase in hospitalization for pneumonia and associated respiratory health ailments (Liu 2013). Asian Sand Dust (ASD), originating from the Gobi Desert and Ocher plateau, is characterized by a median particle size of <10 µm and a defined mineral composition (Honda et al. 2014). The airway epithelium, which is the first barrier to dust and other inhaled particles, responds through multiple pathways, resulting in increased secretion of mucus, involvement of Th2 and Th17 cells, and release of pro-inflammatory cytokines such as IL-6 and IL-8. While this activation is necessary for removal of dust and associated antigens from the respiratory tract; it often results in a pathological immune response leading to inflammatory conditions such as chronic obstructive lung disease (COPD), or exacerbation of seasonal allergies such as rhinitis and pollinosis (Esmaeil et al. 2014). Apart from diseases of the respiratory tract, ASD has been linked to increased incidence of cardiovascular diseases and ischemic strokes in epidemiological studies. In an original article in this issue, Morita and colleagues (Morita et al. 2019) demonstrate that ASD delays the onset of type 1 diabetes in a mouse model, providing links between diabetes and ASD for the first time. Interestingly, adoptively transferred splenocytes from ASD-exposed mice seemed to delay the onset of diabetes in the recipients, suggesting a cell-mediated mechanism with effects on T cell differentiation. This study is yet another example of how complex immune regulation is, and how local modulation can have systemic implications in health and disease.

While extrinsic immunomodulatory particles such as ASD are sporadically and accidentally introduced, intrinsically originating extracellular vesicles are frequently released by cells for the purpose of intercellular communication. There are multiple types of EVs, which are categorized based on size, biogenesis, release pathways and function (Zaborowski et al. 2015). One such category of EVs, called exosomes, are lipid membrane-bound vesicles that are generated and released by the endosomal pathway, ranging in size from 40 to 200 nm. Exosomes are extremely heterogeneous, carrying a slew of proteins, lipids and nucleic acids reflective of their origin (Zhang et al. 2019). The last couple of decades have seen an unprecedented upsurge of interest in exosome research, leading to discoveries of multiple biological functions that are being regulated by exosomes. The heterogeneity of exosomes makes them a double-edged sword with both protective and disease-promoting roles, which are highlighted by three original articles in this issue.

The first article by Matic et al. (Matic et al. 2020) describes the effect of exosomes derived from bovine milk, a common ingredient in our diet, on macrophages, which regulate innate as well as adaptive immune responses. The authors report that these exosomes protect macrophages from death mediated by Cisplatin- a chemotherapeutic agent used in cancer treatment, by arresting their proliferation, providing a classic example of the yin-yang effects of exosomes. The second article by Shenoy et al. (Shenoy et al. 2020) reports a study on exosomes derived from tissues associated with chronic inflammatory diseases such as chronic rhinosinusitis with nasal polyps (CRSwNP) and rheumatoid arthritis. The authors identify exosomes as an immune checkpoint that arrests T cell function, thus possibly contributing to the pathology of these chronic inflammatory diseases. This study also demonstrates that targeting immunosuppressive lipids expressed on the surface of these exosomes may represent a viable strategy to reverse their effects, akin to similar findings in tumor microenvironments (Kelleher et al. 2015; Shenoy et al. 2018). This brings us to the next article, which also describes the immunosuppressive role of exosomes, but in the context of tumors. The article by Shu et al. (Shu et al. 2020) demonstrates the inhibition of tumor antigen-specific T cell activation by exosomes isolated from melanoma cells. The authors also show that blocking the cytokine IL-10, the immune checkpoint PD-L1, or both using monoclonal antibodies can reverse exosome-mediated immunosuppression. These two articles (Shenoy et al. 2020 and Shu et al. 2020), along with many others published before, clearly demonstrate the role of exosomes in disease progression thereby establishing them as a therapeutic target, while offering possible strategies to target them. The article by Shu et al. also showcases advances in technology for the detection and quantification of total and specific subsets of exosomes (such as those expressing PD-L1), which have come a long way since the initial discovery of these vesicles.

Given the roles played by EVs in intercellular communication and immune modulation, together with the advantages associated with their size as well as an absence of self-replicating capabilities, it comes as no surprise that they are being utilized as therapeutic tools to treat a variety of conditions such as microbial infections, neurological disorders and cancer (Gyorgy et al. 2015), and even as drug-delivery vehicles (Elsharkasy et al. 2020). The current issue features a review article by Jafarinia et al. (Jafarinia et al. 2020), focusing on EVs derived from mesenchymal stem cells (MSCs) – the poster child for therapeutic vesicles. In this article, the authors describe the characteristics, isolation methods and biological functions of MSC-EVs, and examine their viability as well as limitations as a potential therapeutic. The review ends with cautious optimism, noting that there is tremendous potential to be harnessed by overcoming the limitations.

The limitations in using EVs as therapeutics are often associated with their heterogeneity, in addition to difficulties in controlling the components and delivery of their cargo. This is overcome by a variety of synthetic and semi-synthetic micro and nano-sized particles that are designed specifically to boost immune responses and fight diseases. Virus-Like Particles (VLP) represent one such category of particles. These are multi-protein structures, ranging in size from 20 to 200 nm, that self-assemble from recombinantly expressed viral structural proteins. VLPs offer unique advantages such as their ability to mimic native viral structure and conformation without carrying the viral genome, uniformity of the assembled particles and constructional flexibility, resulting in safer and cheaper vaccine candidates. Additionally, VLPs are known to be strong activators of the humoral as well as cell-mediated arms of the immune system even in the absence of adjuvants. This has been attributed to the presence of symmetric and repetitive amino acid motifs displayed on their surface that can activate B cell responses; as well as their size, which results in uptake, processing and presentation by APCs, activating T cell responses (Zeltins 2013). These particles have therefore been used as vaccine candidates for more than three decades, and VLPs have been assembled for over 35 families of viruses. While many VLP-based vaccines have been commercialized, others are in clinical trials or undergoing pre-clinical evaluation (Roldao et al. 2010), including candidate vaccines for the devastating global pandemic COVID-19, caused by the coronavirus SARS-CoV-2 (Thanh Le et al. 2020). This issue features an original research article by Lee et al., (Lee et al. 2019) describing a VLP-based vaccine candidate for another pandemic-causing virus – the H1N1 influenza virus, which was responsible for an outbreak in 2009 that lasted for 19 months, resulting in nearly 500,000 cases with more than 18,000 deaths (Low et al., 2010). In this article, the authors compare and report the differences in protective immune responses generated by a candidate VLP vaccine and a commercially available split-virus vaccine. This study showcases some of the advantages of VLP-based vaccines such as the generation of potent immune responses, while highlighting the different factors to be considered for successful vaccine design.

While VLPs can be considered as “natural” or “semi-synthetic” in the sense that they are assembled from naturally occurring viral proteins, a growing list of synthetically generated microspheres and nanoparticles are also being used for immune-modulation. These particles, which may be synthesized from non-biodegradable materials such as metals, or from biodegradable organic components, are utilized to alter the entire spectrum of immune responses from boosting immunity to inducing tolerance. This thematic issue features five articles that focus on different types of synthetic microspheres/nanoparticles and their applications.

The first set of articles describe the use of these particles to boost or elicit robust immune responses. The review article by Crane (Crane 2020) focuses on metal nanoparticles, and provides us with an in-depth look into their potential in treating microbial infections. This article beautifully summarizes the multi-pronged utility of these nanoparticles, discussing their potential as well as the limitations of their potential applications. This is followed by two articles that focus on particles made from biodegradable polymers, which represent a versatile delivery platform as they offer the advantages of being easily surface-modified to enable targeting of specific receptors, and to enable sustained release of encapsulated cargo. The original research article by Han et al. (Han et al. 2020) describes the use of cytokine-encapsulated biodegradable microspheres in anti-tumor immunity. Using fluorescently labeled poly-lactic acid microspheres loaded with IL12, a cytokine that activates T cells (Broderick et al., 2006), the authors demonstrate that intratumorally administered microspheres can drain to the tumor-draining lymph node and elicit anti-tumor responses away from the original site of introduction in a mouse model of cancer. These exciting results once again highlight the advantages of the small size of these particles as well as their immunotherapeutic potential in cancer treatment. The utility of cytokine-encapsulated biodegradable microspheres as immunotherapeutic tools is not limited to cancer; and the entire landscape of its myriad applications is described in the review article by Egilmez (Egilmez 2020). This article offers valuable insights into the use of microspheres formulated with different biodegradable materials, and are encapsulated with a wide variety of cytokines for treating a multitude of conditions such as cancer, infectious diseases, inflammation and degenerative diseases, demonstrating their versatility.

The next set of articles describes the use of synthetic microspheres and nanoparticles to down-modulate unwanted immune responses. The review article by Shahzad et al. (Shahzad et al. 2019) focuses on optimizing the design of biodegradable nanoparticles formulated with polylactic-co-glycolic acid (PLGA) for the purpose of delivering immunomodulatory drugs to prevent allograft rejection. This review highlights the importance of particle design, weighing the pros and cons of different variables such as physicochemical properties, dosage and encapsulation mechanisms in terms of their effect on biological efficacy, including immune modulation. The final article of the thematic issue is a review by Glassman et al. (Glassman et al. 2020) that focuses on the immunosuppressive lipid phosphatidylserine (PS), and describes strategies to harness the ability of PS nanoparticles to tolerize the immune system to its cargo. While replacement therapy with recombinant proteins is often the treatment of choice for diseases such as Hemophilia A and Pompe Disease, immune responses to the administered therapeutic proteins adversely affect their safety and efficacy over time. The authors describe the use of PS nanoparticles to mitigate immunogenicity and tolerize individuals to the otherwise immunogenic protein therapeutics, providing a possible solution to an unmet challenge with widespread implications.

To summarize, this thematic issue features articles across a wide variety of disciplines, and puts the spotlight on microparticles and nanoparticles characterized by their ability to modulate immune responses. We believe this issue celebrates the diversity, versatility and immense potential of these particles, which when present in the right place, at the right time, and in the right amount can either cure diseases or wreak havoc upon the body. We also hope that the articles showcased here pique the readers’ interest and inspire further exploration into the field.