Tamra J. Reed

An Essential Role of Caspase 1 in the Induction of Murine Lupus and Its Associated Vascular Damage

Systemic lupus erythematosus (SLE) is a systemic autoimmune syndrome associated with organ damage and an elevated risk of cardiovascular disease resulting from activation of both innate and adaptive immune pathways. Recently, increased activation of the inflammasome machinery in SLE has been described. Using the mouse model of pristane‐induced lupus, we undertook this study to explore whether caspase 1, the central enzyme of the inflammasome, plays a role in the development of SLE and its associated vascular dysfunction.

Interleukin-5 Supports the Expansion of Fas Ligand-Expressing Killer B Cells that Induce Antigen-Specific Apoptosis of CD4+ T Cells and Secrete Interleukin-10

Beyond their critical role in humoral immunity, B lymphocytes can employ a variety of immunomodulatory mechanisms including expression of the apoptosis-inducing molecule Fas ligand (FasL; CD178). Here, we extensively characterized the surface phenotype of FasL+ killer B cells, showing they are enriched in the IgMhighCD5+CD1dhigh B cell subset previously reported to contain a higher frequency of B cells producing interleukin-10 (IL-10). A rare population of B cells expressing IL-10 was present among FasL+ B cells, but most FasL+ B cells did not produce IL-10. We also identify interleukin-5 (IL-5) as a novel inducer of killer B cell function. Constitutively FasL+ B cells expressed higher levels of the IL-5 receptor, and treating B cells with IL-5 and CD40L resulted in the expansion of a B cell population enriched for FasL+ cells. B cells stimulated with IL-5 and CD40L were potent inducers of apoptosis in activated primary CD4+ T cells, and this killing function was antigen-specific and dependent upon FasL. IL-5 also enhanced IL-10 secretion in B cells stimulated with CD40L. Taken together these findings elucidate the relationship of FasL+ B cells and IL-10-producing B cells and demonstrate that IL-5 can induce or enhance both killer B cell activity and IL-10 secretion in B cells. Finally, we found that the killer B cell activity induced by IL-5 was completely blocked by IL-4, suggesting the existence of a previously unknown antagonistic relationship between these type-2 cytokines in modulating the activity of killer B cells. Targeting this IL-5/IL-4 signaling axis may therefore represent a novel area of drug discovery in inflammatory disorders.

Staphlyococcus aureus Phenol-Soluble Modulins Stimulate the Release of Proinflammatory Cytokines from Keratinocytes and Are Required for Induction of Skin Inflammation

ABSTRACT Staphylococcus aureus is a human commensal that colonizes the skin. While it is normally innocuous, it has strong associations with atopic dermatitis pathogenesis and has become the leading cause of skin and soft tissue infections in the United States. The factors that dictate the role of S. aureus in disease are still being determined. In this work, we utilized primary keratinocyte culture and an epidermal murine colonization model to investigate the role of S. aureus phenol-soluble modulins (PSMs) in proinflammatory cytokine release and inflammation induction. We demonstrated that many species of Staphylococcus are capable of causing release of interleukin 18 (IL-18) from keratinocytes and that S. aureus PSMs are necessary and sufficient to stimulate IL-18 release from keratinocytes independently of caspase 1. Further, after 7 days of epicutaneous exposure to wild-type S. aureus, but not S. aureus Δpsm, we saw dramatic changes in gross pathology, as well as systemic release of proinflammatory cytokines. This work demonstrates the importance of PSM peptides in S. aureus-mediated inflammatory cytokine release from keratinocytes in vitro and in vivo and further implicates PSMs as important contributors to pathogenesis.

Human B Cell-Derived Lymphoblastoid Cell Lines Constitutively Produce Fas Ligand and Secrete MHCII(+)FasL(+) Killer Exosomes.

Immune suppression mediated by exosomes is an emerging concept with potentially immense utility for immunotherapy in a variety of inflammatory contexts, including allogeneic transplantation. Exosomes containing the apoptosis-inducing molecule Fas ligand (FasL) have demonstrated efficacy in inhibiting antigen-specific immune responses upon adoptive transfer in animal models. We report here that a very high frequency of human B cell-derived lymphoblastoid cell lines (LCL) constitutively produce MHCII+FasL+ exosomes that can induce apoptosis in CD4+ T cells. All LCL tested for this study (>20 independent cell lines) showed robust expression of FasL, but had no detectable FasL on the cell surface. Given this intracellular sequestration, we hypothesized that FasL in LCL was retained in the secretory lysosome and secreted via exosomes. Indeed, we found both MHCII and FasL proteins present in LCL-derived exosomes, and using a bead-based exosome capture assay demonstrated the presence of MHCII+FasL+ exosomes among those secreted by LCL. Using two independent experimental approaches, we demonstrated that LCL-derived exosomes were capable of inducing antigen-specific apoptosis in autologous CD4+ T cells. These results suggest that LCL-derived exosomes may present a realistic source of immunosuppressive exosomes that could reduce or eliminate T cell-mediated responses against donor-derived antigens in transplant recipients.

Lupus Skin Is Primed for IL-6 Inflammatory Responses through a Keratinocyte-Mediated Autocrine Type I Interferon Loop

Cutaneous lupus erythematosus is a disfiguring and common manifestation in systemic lupus erythematosus, and the etiology of this predisposition for cutaneous inflammation is unknown. Here, we sought to examine the keratinocyte as an important source of IL-6 and define the mechanism for its increased production in cutaneous lupus erythematosus. Evaluation of discoid and subacute cutaneous lupus erythematosus lesions showed significant epidermal up-regulation of IL-6 compared with control via real-time PCR and immunohistochemistry. Keratinocytes from unaffected skin of lupus patients produced significantly more IL-6 compared with healthy control subjects after exposure to toll-like receptor 2, 3, or 4 agonists or exposure to UVB radiation. Pretreatment with type I interferons (IFN-α and IFN-κ) increased IL-6 production by control keratinocytes, and type I IFN blockade decreased IL-6 secretion by lupus keratinocytes. Secretion of keratinocyte-specific IFN-κ was significantly increased after toll-like receptor 2 and UVB treatment in lupus keratinocytes, and neutralization of IFN-κ decreased IL-6 production by lupus keratinocytes. Thus, lupus keratinocytes are primed for IL-6 hyperproduction in a type I IFN-dependent manner. Increased production of IFN-κ by lupus keratinocytes drives this response, indicating that IFN-κ may play a pathogenic role in cutaneous lupus erythematosus and serve as a target for treatment.

Epidermal injury promotes nephritis flare in lupus-prone mice

Systemic lupus erythematosus is clinically characterized by episodes of flare and remission. In patients, cutaneous exposure to ultraviolet light has been proposed as a flare trigger. However, induction of flare secondary to cutaneous exposure has been difficult to emulate in many murine lupus models. Here, we describe a system in which epidermal injury is able to trigger the development of a lupus nephritis flare in New Zealand Mixed (NZM) 2328 mice. 20-week old NZM2328 female mice underwent removal of the stratum corneum via duct tape, which resulted in rapid onset of proteinuria and death when compared to sham-stripped littermate control NZM2328 mice. This was coupled with a drop in serum C3 concentrations and dsDNA antibody levels and enhanced immune complex deposition in the glomeruli. Recruitment of CD11b(+)CD11c(+)F4/80(high) macrophages and CD11b(+)CD11c(+)F4/80(low) dendritic cells was noted prior to the onset of proteinuria in injured mice. Transcriptional changes within the kidney suggest a burst of type I IFN-mediated and inflammatory signaling which is followed by upregulation of CXCL13 following epidermal injury. Thus, we propose that tape stripping of lupus-prone NZM2328 mice is a novel model of lupus flare induction that will allow for the study of the role of cutaneous inflammation in lupus development and how crosstalk between dermal and systemic immune systems can lead to lupus flare.

Erratum for Syed et al., Staphylococcus aureus Phenol-Soluble Modulins Stimulate the Release of Proinflammatory Cytokines from Keratinocytes and Are Required for Induction of Skin Inflammation

Volume 83, no. 9, p. [3428–3437][1], 2015. Page 3428: The article title should read as given above. Page 3428: The article citation in the footnote block should read as given below. Citation Syed AK, Reed TJ, Clark KL, Boles BR, Kahlenberg JM. 2015. Staphylococcus aureus phenol-soluble modulins

Scleroderma keratinocytes promote fibroblast activation independent of transforming growth factor beta

Objectives SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect. Methods Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-β. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for α-smooth muscle actin (α-SMA), collagen type 1 (COL1A1) and CCL5 expression. Results SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased α-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-β. Microarray analysis identified upregulation of nuclear factor κB (NF-κB) and downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-κB-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes. Conclusion Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-β-independent manner and demonstrate an imbalance in NF-κB1 and PPAR-γ expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy.

2 Shared and endorgan specific transcriptional networks in skin versus kidney biopsies in systemic lupus

Background and aims Skin rash can often herald the onset of a systemic disease flare in systemic lupus. The subtype of skin lesion may confer a differential risk of renal involvement. We hypothesised that renal flares may exhibit crosstalk between skin and kidneys and that similar molecular mechanisms may underlie skin and renal disease. Methods We used systems biology approaches to integrate the regulatory events occurring in subacute cutaneous lupus erythematosus (sCLE, n=43) and discoid lupus erythematosus (DLE, n=47) and compared with those in the ERCB lupus nephritis (LN) class II+IV cohort (n=22). Results Shared transcriptional networks in SLE skin lesions versus LN kidney biopsies reflect similar pathway regulation (p-value<0.05) including complement, B-cells, dendritic cells (DCs), IL4, IL8, and inflammasome signalling pathways. IL-12 signalling and production in macrophages, IL-3, IL-15 signalling pathways were regulated only in LN glomeruli and sCLE rashes, while there were metabolic pathways unique to DLE. CCL21 mRNA expression was specifically up-regulated in sCLE and LN tubulointerstitium and correlated with eGFR, which suggests it may play a role in cutaneous and renal lupus pathogenesis. Conclusions SCLE, which is associated with a higher risk of systemic disease involvement compared with DLE, shares overlapping gene regulation with lupus nephritis. Dendritic cell pathways and associated upregulation of the CCR7 ligand CCL21, that is involved in recruitment of immune effector cells, may serve as a marker for sCLE patients at risk for LN. These data thus identify potentially important molecular targets for novel therapies in cutaneous and renal lupus.

ID: 132: LUPUS KERATINOCYTES ARE PRIMED BY AN AUTOCRINE TYPE I INTERFERON LOOP TO ROBUSTLY SECRETE IL-6

Background Cutaneous lupus erythematosus (CLE) is a disfiguring disease that can affect up to 70% of patients with systemic lupus. Treatment modalities are often ineffective and flares are frequent. Interleukin-6 (IL-6) is a pro-inflammatory cytokine which has gotten recent attention in SLE as IL-6 is increased in the serum of active patients and blockade of IL-6 is therapeutic in murine lupus models and phase I human trials. The source of IL-6 in CLE remains unclear. Methods All studies were approved by the University of Michigan Internal Review Board (IRB# 72843 and 66116 to JMK). RNA was isolated from formalin fixed, paraffin-embedded biopsies of CLE rashes, which were obtained from the University of Michigan Pathology database. Real-time PCR was used to determine the expression level of the myxovirus (influenza virus) resistance 1 (MX-1) and interleukin-6 (IL6) genes. Biopsies were stained for IL-6 using immunohistochemistry. Skin biopsies were obtained from uninvolved skin of SLE patients with a history of cutaneous involvement or healthy controls followed by isolation and culture of keratinocytes. At confluence, cultures were treated with various concentrations of TLR ligands or UVB and IL-6 release was measured via ELISA. Blockade of type I IFN signaling was completed via monoclonal antibody to the type I IFN receptor. Results Real-time PCR analysis of subacute cutaneous lupus erythematosus (sCLE) (n=21) and discoid (DLE) (n=22) rashes demonstrated a significant upregulation of both the IFN-regulated gene, MX1, and the pro-inflammatory cytokine IL-6 when compared with control samples (n=9). Immunohistochemical analysis of skin biopsies confirmed upregulation of IL-6 in the epidermis when compared to control. Keratinocytes from healthy skin of lupus patients produced significantly more IL-6 when stimulated by TLR2, 3 or 4 agonists or exposed to UVB radiation when compared to identical passage keratinocytes from healthy controls. Treatment of control keratinocytes with IFNα increased their IL-6 production and blockade of type I IFNs in the culture media of SKE keratinocytes downregulated the secretion of IL-6. Conclusions IL-6 is increased at the RNA and protein level within cutaneous lupus biopsies when compared to healthy control skin. Keratinocytes are a major producer of IL-6 in the skin and lupus keratinocytes have enhanced production of IL-6 in response to TLR ligands and UV radiation. Exposure to type I IFN can increase IL-6 production in keratinocytes. SLE-derived keratinocytes downregulate IL-6 production in the presence of tonic blockade of the type I IFN receptor. These data suggest that the epidermis, which is an important barrier for environmental insults, is primed for IL-6 production by autocrine type I IFN production and that this may be one mechanism by which factors such as UV exposure may trigger rash development. Further investigations should focus on the pathogenic significance of IL-6 upregulation in the skin and whether targeting this pathway will have an impact on cutaneous disease activity.