Jeroen Deckers

Immunoparalysis is a compensatory and persistent anti-inflammatory response to trauma, sepsis or another serious insult, which increases the risk of opportunistic infections, morbidity and mortality. Here, we show that in cultured primary human monocytes, interleukin-4 (IL4) inhibits acute inflammation, while simultaneously inducing a long-lasting innate immune memory named trained immunity. To take advantage of this paradoxical IL4 feature in vivo, we developed a fusion protein of apolipoprotein A1 (apoA1) and IL4, which integrates into a lipid nanoparticle. In mice and non-human primates, an intravenously injected apoA1-IL4-embedding nanoparticle targets myeloid-cell-rich haematopoietic organs, in particular, the spleen and bone marrow. We subsequently demonstrate that IL4 nanotherapy resolved immunoparalysis in mice with lipopolysaccharide-induced hyperinflammation, as well as in ex vivo human sepsis models and in experimental endotoxemia. Our findings support the translational development of nanoparticle formulations of apoA1-IL4 for the treatment of patients with sepsis at risk of immunoparalysis-induced complications.

Local production of cytokines by infiltrating monocytes/macrophages and Th1 and Th2 cells is of importance in renal allograft rejection. Activated Th1 cells can produce interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and tumor necrosis factor (TNF), whereas Th2 cells produce IL-4 and IL-13, which inhibit Th1 cells. Furthermore, activated T cells express the costimulatory molecule CD40-ligand. During renal allograft rejection, the chemokine RANTES is detected in both infiltrating mononuclear cells and tubular epithelium. It has been shown previously that stimulation of proximal tubular epithelial cells (PTEC) with cytokines or CD40-ligand results in production of RANTES. The present study investigates the influence of Th1 and Th2 cytokines on RANTES production by activated PTEC. RANTES was not detectable in supernatants of human PTEC stimulated with IL-2, IL-4, IL-10, or IL-13 alone. Likewise, combination of these cytokines with IL-1 alpha, IFN-gamma, or TNF-alpha, respectively, did not result in detectable RANTES production. IL-2 and IL-10 had no significant effect on RANTES production by activated PTEC. IL-4 or IL-13 in combination with IL-1 alpha + IFN-gamma or IFN-gamma + TNF-alpha resulted in a two- to fourfold augmentation of RANTES production, ranging from 2.2 +/- 0.2 to 35 +/- 2 ng/ml in different cell lines. CD40-activated PTEC stimulated with IL-4 or IL-13 produced six to ten times more RANTES (ranging from 7.9 +/- 1.9 to 62 +/- 3.5 ng/ml in different cell lines) compared with CD40-activated cells alone. Because RANTES production is augmented by IL-4 and IL-13, this study suggests that during rejection, direct cellular contact between activated Th2 cells and tubular epithelial cells amplifies the local inflammatory reaction in the kidney.

Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.

Interstitial rejection of renal allografts is associated with infiltrating mononuclear cells. Mechanisms leading to this mononuclear cell influx are still not fully resolved. The chemokine RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) is chemotactic for monocytes and T cells. In renal allograft biopsies of patients undergoing rejection, RANTES is found in infiltrating monocytes and T cells, as well as in the tubular epithelium. This study analyzes the production of RANTES in vitro by proximal tubular epithelial cells (PTEC) after stimulation with the inflammatory cytokines interleukin-1alpha, (IL-1alpha), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). Unstimulated PTEC or PTEC stimulated with the cytokines IL-1alpha, IFN-gamma, and TNF-alpha alone did not produce detectable amounts of RANTES. However, a combination of IFN-gamma and either IL-1alpha or TNF-alpha resulted in strong induction of RANTES production up to 2046 +/- 817 pg/ml or 2595 +/- 525 pg/ml per 1 x 10(5) PTEC, respectively. After stimulation with IL-1alpha and TNF-alpha, RANTES production was less prominent than the combination of IFN-gamma with either IL-1alpha or TNF-alpha, and only detectable in 5 of 7 PTEC lines tested. The production of RANTES was both dose- and time-dependent and was inhibited by cycloheximide, indicating that de novo protein synthesis is required. Because the production of RANTES by PTEC is more pronounced in the presence of T cell-derived IFN-gamma (in combination with either IL-1alpha or TNF-alpha), it was hypothesized that RANTES produced by PTEC presumably plays a prominent role in the amplification phase of the immune response rather than in the initiation phase.