Knockdown of the long isoform of the prolactin receptor selectively targets pathogenic immune cells and averts lupus nephritis

Abstract

Abstract Rationale: Systemic lupus erythematosus (SLE) is an autoimmune disease where pathologically activated B and other immune cells induce inflammation that can affect multiple organ systems. Mounting evidence links elevated circulating levels of the pituitary hormone prolactin (PRL) with increased risk of SLE exacerbation. However, mechanisms by which the aberrant expression of specific prolactin receptor (PRLR) isoforms and/or extrapituitary PRL drive autoreactive processes, causing organ damage, remain unknown. Approach: Using high-dimensional flow cytometry and single cell RNA-sequencing, we delineated how PRL, by signaling specifically through the long splice variant of the prolactin receptor (LFPRLR), promotes multiple autoreactive immune cell responses ex vivo in peripheral blood samples from patients with SLE and in vivo in SLE-prone mice. To prevent synthesis of only the LFPRLR isoform in SLE models, we used a patented splice-modulating oligomer (LFPRLR SMO). Results: PRLR expression was aberrantly upregulated in PBMC samples from patients with SLE and in splenocytes during SLE development in primary SLE-prone mice compared to healthy controls. Among PRLR splice isoforms, the anti-apoptotic and pro-proliferative LF, but not the counteractive short isoforms, was aberrantly upregulated in SLE (p=0.007 for human, p<0.0001 for mouse). While cells in every immune subset expressed some PRLR (predominantly LF), classical monocytes and plasma cells were the highest expressers in human PBMCs and mouse splenocytes, respectively. LFPRLR expression in classical monocytes in SLE patient PBMCs positively correlated (p<0.0001) with that of type I interferon (IFN-I) signaling/response genes, known to worsen disease activity in SLE. Thus, we hypothesized LFPRLR may be a therapeutic target in SLE. We measured the effects of a 3-day ex vivo knockdown (KD) of the LFPRLR using the LFPRLR SMO in PBMCs of SLE patients and age- and sex-matched healthy donors. In SLE patient PBMCs, but not in their matched controls, LFPRLR KD significantly reduced pro-inflammatory classical monocytes and CD56-bright NK cells (p<0.05), cell subsets known to drive B-cell activation and self-reactivity. Consistently, LFPRLR KD also reduced B-cell expression of the activation marker CD69, which precedes maturation of B cells into potentially autoreactive memory and plasma cells (p<0.0001). In one sample derived from a patient with multiple autoimmune co-morbidities, LFPRLR KD reduced IFN-I-producing dendritic cell (DC) subsets, atypical B cells, and the ratio of inflammatory Thelper (Th)1/Th17 to regulatory Th2 cell ratio, changes not observed in healthy controls. Therefore, LFPRLR KD partially restores immune homeostasis in SLE. No exogenous human PRL was added while conducting these assays, highlighting the importance of extrapituitary PRL secreted by immune cells in mediating LFPRLR's effects in SLE. To examine the potential long-term benefits of LFPRLR KD, we administered LFPRLR SMO or control SMO for 8 weeks to primary SLE-prone mice in vivo. In the spleen, LFPRLR KD reduced multiple markers of pathologic SLE inflammation: IFN-I signaling and response in immune cells, transcripts of TLR4 pathway genes and CD69 that are indicative of B-cell activation, proliferation and numbers of plasma cells, IFN-I-producing DC subsets, numbers of PRLR+ plasma cells exhibiting a germinal center-like transcriptomic profile, age-associated CD11c+CD11b- B cells, and plasma cells with potentially autoreactive immunoglobulin sequences (p<0.05). In the same mice, we asked whether LFPRLR KD impacts autoantibody-induced glomerulonephritis, characterized by an increase in size, cellularity, and cellular density in the glomerular tufts, the blood filtering units in the kidney. LFPRLR KD averted development of glomerulonephritis such that treated lupus-prone mice resembled healthy mice (p<0.0001). These results demonstrate that LFPRLR KD effectively reduces multiple pathologic immune subsets and prevents kidney damage in an SLE mouse model. Conclusion: Current treatments for SLE lack specific activity against multiple pathogenic innate and adaptive immune subsets. In contrast, LFPRLR SMO reduces only diseased immune cells and averts lupus nephritis. Thus, LFPRLR represents an attractive treatment target in SLE deserving of further clinical development.