Investigating the role of interleukin-4 and type 2 immune responses in articular joint health and disease
Keywords: Stem Cell Biology and Regenerative Medicine.

Osteoarthritis (OA), often called degenerative joint disease, is widely accepted as the most common type of joint disease[1], affecting 14% of adults, over 30 million individuals, in the U.S. annually from 2008-2014. Despite the high prevalence, significant economic burden, and debilitating impact on individuals with OA, there currently are no disease modifying treatments available for OA. Current therapeutic strategy is largely palliative and focuses on symptom management. For end-stage disease in patients with severe functional impairment, total joint replacement is considered both clinically-relevant and cost-effective[2]. While the current therapeutic strategies are effective for symptom management, the focus of drug development has shifted to disease-modifying agents, in an effort to more effectively address the larger epidemic of OA. Historically, OA has been thought of as a simple 'wear-and-tear' mechanically-driven disease. In the past decade, improved understanding of OA pathobiology has challenged this view, instead favoring the view of OA as a multifactorial disorder with many significant drivers of disease. Identification and characterization of these additional 'significant drivers' has and will continue to identify new targets for therapeutic intervention. Though OA is not thought of as classical "inflammatory arthritis", chronic, low-grade inflammation is now thought to be one of the significant drivers of disease, playing a central role in pathogenesis[3]. The current understanding of the role of inflammation in OA is that injury (acute, subacute, or chronic) initiates tissue damage that, in turn, triggers an inflammatory response. In the context of other risk factors such as obesity, aging, or other genetic factors, this sequence of events can trigger a vicious cycle of inflammatory response to local tissue damage and failed tissue regeneration, ultimately leading to joint degeneration[3]. One inflammatory pathway that has been less commonly studied in OA is that of interleukin-4 (IL-4), an important cytokine in type 2 immunity. Though yet to be understood as a driver of disease, IL-4 pathway components have been linked to OA with genomic analyses identifying associations between IL-4R polymorphisms and hand OA[4], knee OA[5], and hip OA[6]. Additionally, a number of joint-resident cell types are known to be IL-4-responsive, further supporting the hypothesis that IL-4 may play an important role in joint homeostasis. In this thesis, I attempt to demonstrate my overarching hypothesis that, though OA is not a disease of type 2 immunity, components of type 2 immune responses can contribute to maintenance of joint health and protect against OA. In Chapter 2, we investigated the role of IL-4 in OA pathogenesis using in vivo and in vitro models of disease. Mice deficient in various IL-4 pathway components were subjected to destabilization of the medial meniscus to induce OA. Macrophages, osteoclasts, and synovial explants were stimulated with IL-4 in vitro, and their function and expression profiles characterized. We found that mice lacking in IL-4 cytokine, receptors, and signal transducers developed exacerbated cartilage damage and osteophyte formation relative to WT controls. In vitro analyses revealed that IL-4 downregulates osteoarthritis-associated genes, enhances macrophage phagocytosis of cartilage debris, and inhibits osteoclast differentiation and activation via the type I receptor. Taken together, our findings demonstrate that IL-4 protects against osteoarthritis in a myeloid and STAT6-dependent manner. Further, IL-4 can promote an immunomodulatory microenvironment in which joint-resident macrophages polarize towards an M2 phenotype and efficiently clear pro-inflammatory debris, and osteoclasts maintain a homeostatic level of activity in subchondral bone. These findings support a role for IL-4 modulation of myeloid cell types in maintenance of joint health and identify a pathway that could provide therapeutic benefit for osteoarthritis. In Chapter 3, we applied whole-transcriptomic approaches to characterize the effect of IL-4 on osteoclasts, an IL-4 responsive joint-resident cell that is dysregulated in OA and compared this effect to that on macrophages. In this study, unstimulated and IL-4-stimulated murine bone marrow-derived macrophages and osteoclast precursors were profiled by RNA sequencing analysis and the resulting expression patterns compared by DESeq2 and Sdef analysis. Principal component analysis revealed that IL-4 stimulated OCPs are transcriptionally distinct, and Sdef and GSEA identified enrichment of both shared and unique pathways, supporting the hypothesis that the IL-4-stimulated state in OCs resembles, but is distinct from the IL-4-stimulated state in macrophages. This study reported the first application of whole-transcriptomic analysis of the effects of IL-4 on osteoclasts. These studies used numerous methods to investigate the overarching hypothesis that the Type 2 immunity cytokine IL-4 plays a role in pathogenesis of OA. We demonstrate, in vivo, the protective role of IL-4 against the development of post-traumatic OA via its effects on myeloid cells. Additionally, we report the first transcriptomic level characterization of the effect of IL-4 on osteoclast differentiation and activation. We believe that these findings highlight the IL-4 pathway potentially therapeutically relevant for the treatment of OA. Strategies such as the use of targeted superkines[7] to supplement IL-4 in the joint may provide insights into new ways of treating OA. Future work related to this discovery will focus on the investigation of the therapeutic use of IL-4, in a mouse model of OA and additional efforts to further implicate IL-4 signaling in human musculoskeletal tissues. References 1. Kumar V, Abbas AK, Aster JC, Perkins JA. Robbins and Cotran Pathologic Basis of Disease. In. Ninth edition ed. Philadelphia, PA: Elsevier/Saunders; 2015. 2. Hunter DJ, Bierma-Zeinstra S. Osteoarthritis. The Lancet. 2019; 393(10182):1745-1759. 3. Robinson WH, Lepus CM, Wang Q, et al. Low-grade inflammation as a key mediator of the pathogenesis of osteoarthritis. In. Vol 122016:580-592. 4. Vargiolu M, Silvestri Z A T, Bonora E, et al. Interleukin-4/interleukin-4 receptor gene polymorphisms in hand osteoarthritis. Osteoarthritis and Cartilage. 2010; 18:810-816. 5. Rogoveanu OC, Calina D, Cucu MG, et al. Association of cytokine gene polymorphisms with osteoarthritis susceptibility. Exp Ther Med. 2018; 16(3):2659-2664. 6. Forster T, Chapman K, Loughlin J. Common variants within the interleukin 4 receptor ? gene (IL4R) are associated with susceptibility to osteoarthritis. Human Genetics. 2004; 114(4):391-395. 7. Junttila IS, Creusot RJ, Moraga I, et al. Redirecting cell-type specific cytokine responses with engineered interleukin-4 superkines. Nat Chem Biol. 2012; 8(12):990-998