Cytokines are fundamental components of the immune system and understanding their biology is a necessary step in understanding immune functions in both health and disease. Cytokines represent key mediators of the immune system which act as molecular messages that regulate both the initiation and the phenotype of the immune responses. Because of their central role in shaping immune functions, every new cytokine that has been reported has generated significant interest and has created a new field of research.

Here, I describe the identification of a novel cytokine Metrnl, which we proposed to be renamed interleukin-39 (IL-39) to reflect its association with functions of the immune system. I have explored in-depth regulation of IL-39 expression in macrophages and found that its production is regulated by multiple stimuli. Specifically, IL-39 production is highly induced by several cytokines including TNFα, IL-17, IL-12 and IL-4 whereas TGFβ and IFNƴ inhibit its production. Additionally, I have found that physiological levels of IL-39 significantly increase in response to sterile inflammation induced by thioglycollate injection. To elucidate function(s) of IL-39, I have generated a colony of IL-39-/- mice. Initial phenotyping of these mice did not reveal defects in immune cell populations in major immune compartments such as in the thymus, spleen, lymph nodes and bone marrow. Many cytokines such as IFNƴ, IL-4, TGFβ and IL-10 play a role in humoral immune responses. Therefore, I compared serum IgM, total IgG and IgA levels in IL-39 deficient and wild type mice and found that IL-39-/- mice had increased levels of IgM and decreased levels of total IgG. Among four IgG isotypes, I found significant reduction in IgG3 and IgG2b in serum of IL-39 deficient mice. Furthermore, splenocytes isolated from IL-39-/- mice produced altered levels of several cytokines and chemokines under T-cell activation conditions (with anti-CD3/anti-CD28). For example, splenocytes from IL-39 deficient mice secreted increased amount of CXCL9, CXCL10 and IL-2 while the levels of CCL3, CCL4 and IFNƴ were reduced. Because these cytokines and chemokines play important roles in protection against many pathogens, we sought to determine if IL-39 plays a role in immune response to a murine model of a T.gondii infection. Indeed, we found that IL-39-/- mice were more susceptible to the infection and displayed altered levels of several immune cell populations. This includes reduced levels of CD4+ T cells and CD8+ T cells in spleens of IL-39-/- mice when compared to WT controls. Together, these results indicate that IL-39 is a cytokine that is produced by macrophages in response to inflammation and has important functions in regulating innate and/or adaptive immune responses.

We describe a novel B cell-associated cytokine, encoded by an uncharacterized gene (C17orf99), whose expression is induced in B cells upon activation. C17orf99 is only present in mammalian genomes, and it encodes a small (~27kDa) secreted protein unrelated to other cytokine families, suggesting a function in mammalian immune responses. Accordingly, C17orf99 expression is induced in the mammary gland upon the onset of lactation, and a C17orf99-/- mouse exhibits reduced levels of IgA in the serum, gut, feces, and lactating mammary gland. C17orf99-/- mice have smaller and fewer Peyer’s patches and lower numbers of IgA secreting cells. The microbiome of C17orf99-/- mice exhibits altered composition, likely a consequence of the reduced levels of IgA in the gut. While naïve B cells can express C17orf99 upon activation, their production increases following culture with various cytokines, including IL4 and TGF-β, suggesting that differentiation can result in the expansion of C17orf99-producing B cells during some immune responses. Taken together, these observations indicate that C17orf99 encodes a novel B cell-associated cytokine, which we have called Interleukin-40, that plays an important role in the development of humoral immune responses. Importantly, IL-40 is also expressed by human activated B cells and by several human B cell lymphomas. The latter observation suggests that it may play a role in the pathogenesis of certain human diseases.

Immune responses are regulated by an intricate balance between cytokine production and actions of various immune cell types. Cytokines are key regulators of the immune system and represent molecular messengers that provide extrinsic cues that direct the genesis of an immune response and its ultimate down-regulation. Here, I aim to characterize two cytokines that play distinct roles in the development of humoral immune responses.

First, I describe a novel B cell cytokine, Interleukin 40 (IL-40) that is expressed in hematopoetic tissues (fetal liver and bone marrow) and by a lymphocyte population not regularly associated with cytokine secretion (activated B cells). Interestingly, the IL-40 gene is only present in mammalian genomes, indicating that it may participate in a mammalian-specific immune function. One such mammalian specific function which is also immune related is the process of lactation. The mammary gland undergoes extensive remodeling upon the onset of lactation. Milk ducts become defined to transport milk and the immune microenvironment undergoes significant changes that lead to the production of IgA. Importantly, IL-40 is up-regulated immediate after lactation. An IL-40 deficient mouse (Il-40-/- mouse), exhibits an altered B cell phenotype, reduced serum and mucosal IgA, and a blunted response to HI-VACV (heat-inactivated vaccinia virus). I next focus on CCL28, a mucosal chemotactic cytokine. CCL28 is responsible for the recruitment of IgA secreting cells to mucosal sites via its receptor CCR10. Additionally, CCL28 is a potent antimicrobial peptide that can inhibit gram positive/gram negative bacteria, and C. albicans. Here, I functionally characterize the first mouse with a targeted deletion of the Ccl28 gene, (Ccl28-/- mouse). The Ccl28-/- mouse exhibits altered levels of IgA in mucosal secretions, and high susceptibility to salmonella infection.

Overall, I have functionally characterized two distinct cytokine knock-out mouse models, IL-40 and CCL28. I predict that through my doctoral work I have laid the foundation for a better understanding of the role that these two cytokines play in the fields of B cell biology, cytokine discovery, mucosal immunology and the pathogenesis of mucosal infections.

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