Pivotal role of linear ubiquitin mediator SHARPIN in dendritic cell homeostasis and function in mice

Zhe Wang, Purdue University

Abstract

SHANK-associated RH domain interacting protein (SHARPIN) is a novel protein of which the physiological functions are just beginning to be understood. Two allelic, loss-of-expression mutations occurred spontaneously in mice ( Sharpincpdm mice), resulting in chronic dermatitis, multiorgan inflammation and disrupted development of lymphoid tissues, suggesting a pivotal role for SHARPIN in regulating immune homeostasis in steady-state conditions. The chronic skin inflammation is characterized by upregulated expression of type 2 cytokines and massive infiltration of eosinophils, resembling aspects of atopic dermatitis and hypereosinophilic syndrome in humans. The pathogenic mechanism by which the inflammatory disease develops remains poorly defined. Recent studies have provided important molecular insights into the role of SHARPIN in modulating inflammation and immunity. SHARPIN is a structural component of the linear ubiquitin assembly complex (LUBAC), and regulates the activation of inflammatory signaling pathways, in particular nuclear factor k light chain in B cells (NF-κB), by conjugating and activating target proteins, such as NF-κB essential modulator (NEMO). In addition, SHARPIN is involved in modulating cellular susceptibility to tumor necrosis factor (TNF)-induced apoptosis, as Sharpin-deficient mouse embryo fibroblasts become highly sensitive to cell death. Indeed, an increased number of dead keratinocytes is present in the skin of Sharpincpdm mice, and it has been proposed that the TNF-induced keratinocyte cell death initiates the pathogenesis of the dermatitis. However, the underlying pathogenic process is still poorly understood. Dendritic cells (DCs) are an important type of innate cells in the frontline of immune defense. They effectively detect microbial invasion, environmental insults and presence of cell injury or death, and rapidly activate innate immunity. DCs are also important for initiating and directing acquired immune responses by secreting immunoregulatory factors, such as cytokines. Altered functions of DCs can result in inflammatory disorders as demonstrated in mouse models. Given the dysregulated cytokine profile and presence of apoptotic keratinocytes in Sharpincpdm mice, I hypothesized that SHARPIN is a novel regulator of DC immune functions and DC-specific SHARPIN expression is critical for maintaining immune homeostasis. The present study has three aims. First, the immune functions of DCs were examined in the absence of Sharpin expression, including cytokine secretion, activation of inflammatory signaling and T-cell stimulating capacity. Second, activation of inflammasomes and IL1κ production were determined in Sharpincpdm mice, as interference with signaling by cytokines that belong to the IL1 family improved the skin disease. Last, the DC-intrinsic role was investigated by genetically ablating Sharpin specifically in DCs, and determining the effect on immune functions. Our results demonstrated that Sharpin deficiency in bone marrow-derived DCs (BMDCs) resulted in selective defects in NF-κB activation and impaired secretion of inflammatory cytokines induced by TOLL-like receptor (TLR) agonists LPS and poly I:C. Furthermore, absence of Sharpin expression interfered with the ability of BMDCs to induce differentiation of Th1 CD4 T cells. The dermatitis in Sharpin cpdm mice is associated with upregulated IL1κ expression at both mRNA and protein levels. This possibly results from aberrant activation of the NRLP1 inflammasome, as BMDCs from Sharpincpdm mice exhibited enhanced IL1κ production in response to NLRP1-specific activators, anthrax lethal toxin (LT). Intriguingly, supernatants from keratinocytes that were stimulated with TNF for 24 hours also induced IL1κ secretion from WT BMDCs and more robustly from Sharpin-deficient BMDCs. Transgenic mice lacking Sharpin specifically in DC-lineage cells were generated. It was found that these conditional knockout mice had impaired antigen-specific immune response in immunization studies. Collectively, the present study indicates that DC-specific SHARPIN expression is indispensible for maintaining full strength of immune function and regulating in vivo immunity to antigen challenge.

Degree

Ph.D.

Advisors

Esch, Purdue University.

Subject Area

Biology|Cellular biology|Immunology

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