Fibroblasts are stromal cells known to regulate local immune responses important for wound healing and scar formation; however, the cellular mechanisms driving damage and scarring in cutaneous lupus erythematosus (CLE) patients remain poorly understood. Dermal fibroblasts in systemic lupus erythematosus (SLE) patients are abnormally exposed to cytokines, but the impact of inflammatory mediators on fibroblast responses in non-scarring versus scarring CLE subtypes is unclear. Here, we examined responses to cytokines in dermal fibroblasts from non-lesional skin of 22 SLE patients with CLE and 34 healthy controls. Notably, inflammatory cytokine responses were exaggerated in SLE fibroblasts compared to healthy controls. In lesional CLE biopsies, these same inflammatory profiles were reflected in single cell RNA sequencing of SFRP2+ and inflammatory fibroblast subsets, and TGF-β was identified as a critical upstream regulator for inflammatory fibroblasts in scarring discoid lupus lesions. In vitro cytokine stimulation of non-lesional fibroblasts from patients who scar from CLE identified an upregulation of collagens, particularly in response to TGF-β, whereas inflammatory pathways were more prominent in non-scarring patients. Our study revealed that SLE fibroblasts are poised to hyper-respond to inflammation, with differential responses among scarring versus non-scarring disease, providing a potential skin-specific target for mitigating damage.
Suzanne K. Shoffner-Beck, Lisa Abernathy-Close, Stephanie Lazar, Feiyang Ma, Mehrnaz Gharaee-Kermani, Amy Hurst, Craig Dobry, Deepika Pandian, Rachael Wasikowski, Amanda Victory, Kelly Arnold, Johann E. Gudjonsson, Lam C. Tsoi, J. Michelle Kahlenberg
Linear ubiquitin chains, which are generated specifically by the linear ubiquitin assembly complex (LUBAC) ubiquitin ligase, play crucial roles in immune signaling, including NF-κB activation. LUBAC comprises catalytic large isoform of heme-oxidized iron regulatory protein 2 ubiquitin ligase 1 (HOIL-1L) interacting protein (HOIP), accessory HOIL-1L, and SHANK-associated RH domain-interacting protein (SHARPIN). Deletion of the ubiquitin ligase activity of HOIL-1L, an accessory ligase of LUBAC, augments LUBAC functions by enhancing LUBAC-mediated linear ubiquitination, which is catalyzed by HOIP. Here, we show that HOIL-1L ΔRING1 mice, which exhibit augmented LUBAC functions upon loss of the HOIL-1L ligase, developed systemic lupus erythematosus (SLE) and Sjögren’s syndrome in a female-dominant fashion. Augmented LUBAC activity led to hyperactivation of both lymphoid and myeloid cells. In line with the findings in mice, we sought to identify missense single nucleotide polymorphisms/variations of the RBCK1/HOIL-1L gene in humans that attenuate HOIL-1L ligase activity. We found that the R464H variant, which is encoded by rs774507518 within the RBCK1/HOIL-1L gene, attenuated HOIL-1L ligase activity and augmented LUBAC-mediated immune signaling, including that mediated by Toll-like receptors. We also found that rs774507518 was enriched significantly in patients with SLE, strongly suggesting that RBCK1/HOIL-1L is an SLE susceptibility gene and that augmented linear ubiquitin signaling generated specifically by LUBAC underlies the pathogenesis of this prototype systemic autoimmune disease.
Yasuhiro Fuseya, Keiichiro Kadoba, Xiaoxi Liu, Hiroyuki Suetsugu, Takeshi Iwasaki, Koichiro Ohmura, Takayuki Sumida, Yuta Kochi, Akio Morinobu, Chikashi Terao, Kazuhiro Iwai
Lupus nephritis (LN) is a pathologically heterogenous autoimmune disease linked to end-stage kidney disease and mortality. Better therapeutic strategies are needed as only 30%–40% of patients completely respond to treatment. Noninvasive biomarkers of intrarenal inflammation may guide more precise approaches. Because urine collects the byproducts of kidney inflammation, we studied the urine proteomic profiles of 225 patients with LN (573 samples) in the longitudinal Accelerating Medicines Partnership in RA/SLE cohort. Urinary biomarkers of monocyte/neutrophil degranulation (i.e., PR3, S100A8, azurocidin, catalase, cathepsins, MMP8), macrophage activation (i.e., CD163, CD206, galectin-1), wound healing/matrix degradation (i.e., nidogen-1, decorin), and IL-16 characterized the aggressive proliferative LN classes and significantly correlated with histological activity. A decline of these biomarkers after 3 months of treatment predicted the 1-year response more robustly than proteinuria, the standard of care (AUC: CD206 0.91, EGFR 0.9, CD163 0.89, proteinuria 0.8). Candidate biomarkers were validated and provide potentially treatable targets. We propose these biomarkers of intrarenal immunological activity as noninvasive tools to diagnose LN and guide treatment and as surrogate endpoints for clinical trials. These findings provide insights into the processes involved in LN activity. This data set is a public resource to generate and test hypotheses and validate biomarkers.
Andrea Fava, Jill Buyon, Laurence Magder, Jeff Hodgin, Avi Rosenberg, Dawit S. Demeke, Deepak A. Rao, Arnon Arazi, Alessandra Ida Celia, Chaim Putterman, Jennifer H. Anolik, Jennifer Barnas, Maria Dall’Era, David Wofsy, Richard Furie, Diane Kamen, Kenneth Kalunian, Judith A. James, Joel Guthridge, Mohamed G. Atta, Jose Monroy Trujillo, Derek Fine, Robert Clancy, H. Michael Belmont, Peter Izmirly, William Apruzzese, Daniel Goldman, Celine C. Berthier, Paul Hoover, Nir Hacohen, Soumya Raychaudhuri, Anne Davidson, Betty Diamond, the Accelerating Medicines Partnership in RA/SLE network, Michelle Petri
Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This library was validated using seven genetic mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2–/– and µMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn–/– IgD+/– mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire–/– mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.
Elze Rackaityte, Irina Proekt, Haleigh S. Miller, Akshaya Ramesh, Jeremy F. Brooks, Andrew F. Kung, Caleigh Mandel-Brehm, David J.L. Yu, Colin R. Zamecnik, Rebecca Bair, Sara E. Vazquez, Sara Sunshine, Clare L. Abram, Clifford A. Lowell, Gabrielle Rizzuto, Michael R. Wilson, Julie Zikherman, Mark S. Anderson, Joseph L. DeRisi
The selective targeting of pathogenic T cells is a “holy grail” in the development of new therapeutics for T cell-mediated disorders including many autoimmune diseases and graft-versus-host disease. We describe the development of a CD6-targeted antibody-drug conjugate (CD6-ADC) by conjugating an inactive form of monomethyl auristatin E (MMAE), a potent mitotic toxin, onto a monoclonal antibody (mAb) against CD6, an established T cell surface marker. Even though CD6 is present on all T cells, only the activated (pathogenic) T cells vigorously divide and thus are susceptible to the anti-mitotic MMAE-mediated killing via the CD6-ADC. We found CD6-ADC selectively killed activated proliferating human T cells and antigen-specific mouse T cells in vitro. Furthermore, in vivo, whereas the CD6-ADC had no significant detrimental effect on normal T cells in naïve CD6 humanized mice, the same dose of CD6-ADC, but not the controls, efficiently treated two pre-clinical models of autoimmune uveitis and a model of graft-versus-host disease. These results provide evidence suggesting that CD6-ADC could be further developed as a novel therapeutic agent for the selective elimination of pathogenic T cells and treatment of many T cell-mediated disorders.
Lingjun Zhang, Liping Luo, Jin Y. Chen, Rupesh Singh, William M. Baldwin III, David A. Fox, Daniel J. Lindner, Daniel F. Martin, Rachel R. Caspi, Feng Lin
Variation in the preservation of β cell function in clinical trials in type 1 diabetes (T1D) has emphasized the need to define biomarkers to predict treatment response. The T1DAL trial targeted T cells with alefacept (LFA-3-Ig) and demonstrated C-peptide preservation in ~30% of new onset T1D subjects. We analyzed islet antigen reactive (IAR) CD4 T cells in PBMC samples collected prior to treatment from alefacept- and placebo-treated subjects using flow cytometry and single cell RNA-sequencing. IAR CD4 T cells at baseline had heterogenous phenotypes. Transcript profiles formed phenotypic clusters of cells along a trajectory based on increasing maturation and activation, and T cell receptor (TCR) chains showed clonal expansion. Notably, the frequency of IAR CD4 T cells with a memory phenotype and a unique transcript profile (Cluster 3) were inversely correlated with C-peptide preservation in alefacept-, but not placebo-treated subjects. Cluster 3 cells had a proinflammatory phenotype characterized by expression of the transcription factor BHLHE40 and the cytokines GM-CSF and TNF-α, and shared TCR chains with effector memory-like clusters. Our results suggest IAR CD4 T cells as a potential baseline biomarker of response to therapies targeting the CD2 pathway and warrant investigation for other T cell-related therapies.
Elisa Balmas, Janice Chen, Alex K. Hu, Hannah A. DeBerg, Mario G. Rosasco, Vivian H. Gersuk, Elisavet Serti, Cate Speake, Carla J. Greenbaum, Gerald T. Nepom, Peter S. Linsley, Karen Cerosaletti
We previously reported that treatment of mice with 6-gingerol, the most abundant phytochemical in ginger root, leads to phosphodiesterase inhibition that counteracts neutrophil hyperactivity in models of antiphospholipid syndrome (APS) and lupus. Here, we explored the extent to which oral intake of a whole-ginger extract would similarly impact neutrophils in both autoimmune mice and healthy humans. In vitro, a solubilized ginger extract was able to attenuate neutrophil extracellular trap formation (NETosis) by human neutrophils through a mechanism that was dependent upon the cyclic AMP–dependent kinase, protein kinase A. When mice with features of either APS or lupus were administered a ginger extract orally, they demonstrated reduced circulating NETs, as well as the tempering of other disease outcomes, such as large-vein thrombosis (APS) and autoantibody production (lupus). In a pilot clinical trial, which was validated in a second cohort, daily intake of a ginger supplement for 7 days by healthy volunteers boosted neutrophil cAMP, inhibited NETosis in response to disease-relevant stimuli, and reduced circulating plasma NET levels. In summary, this work demonstrates that ginger intake restrains neutrophil hyperactivity in autoimmune mouse models and that ginger consumption by healthy individuals makes their neutrophils more resistant to NETosis.
Ramadan A. Ali, Valerie C. Minarchick, Miela Zahavi, Christine E. Rysenga, Kristin A. Sturm, Claire K. Hoy, Cyrus Sarosh, Jason S. Knight, M. Kristen Demoruelle
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a dramatic sex bias, affecting 9-times more women than men. Activation of toll-like receptor 7 (TLR7) by self-RNA is a central pathogenic process leading to aberrant production of type-I interferon (IFN) in SLE, but the specific RNA molecules that serve as TLR7 ligands have not been defined. We therefore sought to identify female-specific endogenous RNAs containing canonical TLR7 stimulatory motifs. By leveraging gene expression data and the known sequence specificity of TLR7, we identified the female-specific X-inactive specific transcript (XIST) long non-coding RNA as a uniquely rich source of TLR7 ligands in SLE. XIST RNA stimulated IFNα production by plasmacytoid DCs in a TLR7-dependent manner, and deletion of XIST diminished the ability of whole cellular RNA to activate TLR7. XIST levels were elevated in blood leukocytes from female SLE patients compared to controls, correlated positively with disease activity and the IFN signature, and were enriched in extracellular vesicles released from dying cells in vitro. Importantly, XIST was not IFN-inducible, suggesting that XIST is a driver, rather than a consequence of IFN in SLE. Our work suggests a novel role for XIST RNA as a female-specific danger signal underlying the sex bias in SLE.
Jonathan D. Crawford, Hong Wang, Daniela Trejo-Zambrano, Raffaello Cimbro, C. Conover Talbot Jr., Mekha A. Thomas, Ashley M. Curran, Alexander A. Girgis, John T. Schroeder, Andrea Fava, Daniel W. Goldman, Michelle Petri, Antony Rosen, Brendan Antiochos, Erika Darrah
Abnormal macrophage polarization is generally present in autoimmune diseases. Overwhelming M1 macrophage activation promotes the continuous progression of inflammation, which is one of the vital reasons for the development of autoimmune diseases. However, the underlying mechanism is still unclear. Here we explore the function of RFX1 in macrophage polarization by constructing colitis and lupus-like mouse models. Both in vivo and in vitro experiments confirmed that RFX1 can promote M1 and inhibit M2 macrophage polarization. Besides, we also found that RFX1 promoted DNA demethylation of macrophage polarization-related genes by increasing APOBEC3A/Apobec3 expression. Noteworthily, we identified a potential RFX1 inhibitor, adenosine diphosphate (ADP), providing a potential strategy for treating autoimmune diseases.
Shuang Yang, Pei Du, Haobo Cui, Meiling Zheng, Wei He, Xiaofei Gao, Zhi Hu, Sujie Jia, Qianjin Lu, Ming Zhao
Many autoimmune diseases (AIDs) are characterized by persistence of autoreactive B cell responses which is often directly implicated in disease pathogenesis. How and why these cells are generated or how they are maintained for years is largely unknown. Rheumatoid arthritis is among the most common AIDs and characterized by autoantibodies recognizing proteins with post-translational modifications (PTMs). This PTM-directed, autoreactive B cell compartment is ill defined. Here, we visualized the B cell response against the three main types of PTM antigens implicated in RA by spectral flow cytometry. Our results showed extensive cross-reactivity of PTM-directed B cells against all three PTM antigens (citrulline, homocitrulline and acetyllysine). Unsupervised clustering revealed several distinct memory B cell (mBC) populations. PTM-directed cells clustered with the most recently activated, class-switched mBC phenotype, expressing high CD80, low CD24 and low CD21. Notably, patients also harbored large fractions of PTM-directed plasmablasts (PB). Both PTM-directed mBC and PB showed high expression of CXCR3, a receptor for chemokines abundantly present in arthritic joints. Together, our data provide detailed insight into the biology of B cell autoreactivity and its remarkable, seemingly exhaustless persistence in a prominent human AID.
Sanne Reijm, Joanneke C. Kwekkeboom, Nienke J. Blomberg, Jolien Suurmond, Diane Van der Woude, Rene E.M. Toes, Hans Ulrich Scherer
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