The mRNA-Binding Protein KSRP Limits the Inflammatory Response of Macrophages.

KH-type splicing regulatory protein (KSRP) is a single-stranded nucleic acid-binding protein with multiple functions. It is known to bind AU-rich motifs within the 3'-untranslated region of mRNA species, which in many cases encode dynamically regulated proteins like cytokines. In the present study, we investigated the role of KSRP for the immunophenotype of macrophages using bone marrow-derived macrophages (BMDM) from wild-type (WT) and KSRP-/- mice. RNA sequencing revealed that KSRP-/- BMDM displayed significantly higher mRNA expression levels of genes involved in inflammatory and immune responses, particularly type I interferon responses, following LPS stimulation. In line, time kinetics studies revealed increased levels of interferon-γ (IFN-γ), interleukin (IL)-1ß and IL-6 mRNA in KSRP-/- macrophages after 6 h subsequent to LPS stimulation as compared to WT cultures. At the protein level, KSRP-/- BMDM displayed higher levels of these cytokines after overnight stimulation. Matching results were observed for primary peritoneal macrophages of KSRP-/- mice. These showed higher IL-6, tumor necrosis factor-α (TNF-α), C-X-C motif chemokine 1 (CXCL1) and CC-chemokine ligand 5 (CCL5) protein levels in response to LPS stimulation than the WT controls. As macrophages play a key role in sepsis, the in vivo relevance of KSRP deficiency for cytokine/chemokine production was analyzed in an acute inflammation model. In agreement with our in vitro findings, KSRP-deficient animals showed higher cytokine production upon LPS administration in comparison to WT mice. Taken together, these findings demonstrate that KSRP constitutes an important negative regulator of cytokine expression in macrophages.

Nanobodies Outperform Antibodies - Rapid Functionalization with Equal In Vivo Targeting Properties.

Highly specific targeting of dendritic cells in vivo is crucial for the development of effective tumor nanovaccines. This group recently presented an antibody-functionalized nanocarrier system able to maintain its targeting properties when transferred from in vitro to in vivo studies. However, producing this system requires long synthesis times and involves high expenses due to the involved site-specific enzymatic multi-step modification procedure of the antibody. Consequently, improving the previously proposed system is necessary in order to accelerate the development. Here, a novel system utilizing nanobodies for the targeting of dendritic cells is presented. A C-terminal cysteine tag facilitates an easy attachment of the nanobody to the nanocarrier via a thiol-maleimide conjugation technique. This reduces the functionalization time from several days to mere hours. Using in vitro and in vivo assays, it is shown that the optimized system possesses equal targeting properties as the antibody-based system. As a result, nanobodies and the coupling chemistry are found to be a superior strategy for the in vivo targeting of dendritic cells when compared to antibodies, due to their rapid attachment to nanocarriers and equal targeting specificity. This would replace antibodies as the current "gold standard" of targeting moieties.

Multicompartment Polyion Complex Micelles Based on Triblock Polypept(o)ides Mediate Efficient siRNA Delivery to Cancer-Associated Fibroblasts for Antistromal Therapy of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and the third leading cause for cancer-related death worldwide. The tumor is difficult-to-treat due to its inherent resistance to chemotherapy. Antistromal therapy is a novel therapeutic approach, targeting cancer-associated fibroblasts (CAF) in the tumor microenvironment. CAF-derived microfibrillar-associated protein 5 (MFAP-5) is identified as a novel target for antistromal therapy of HCC with high translational relevance. Biocompatible polypept(o)ide-based polyion complex micelles (PICMs) constructed with a triblock copolymer composed of a cationic poly(l-lysine) complexing anti-MFAP-5 siRNA (siMFAP-5) via electrostatic interaction, a poly(γ-benzyl-l-glutamate) block loading cationic amphiphilic drug desloratatine (DES) via π-π interaction as endosomal escape enhancer and polysarcosine poly(N-methylglycine) for introducing stealth properties, are generated for siRNA delivery. Intravenous injection of siMFAP-5/DES PICMs significantly reduces the hepatic tumor burden in a syngeneic implantation model of HCC, with a superior MFAP-5 knockdown effect over siMFAP-5 PICMs or lipid nanoparticles. Transcriptome and histological analysis reveal that MFAP-5 knockdown inhibited CAF-related tumor vascularization, suggesting the anti-angiogenic effect of RNA interference therapy. In conclusion, multicompartment PICMs combining siMFAP-5 and DES in a single polypept(o)ide micelle induce a specific knockdown of MFAP-5 and demonstrate a potent antitumor efficacy (80% reduced tumor burden vs untreated control) in a clinically relevant HCC model.

Topical Application of Adenosine A2-Type Receptor Agonists Prevents Contact Hypersensitivity Reactions in Mice by Affecting Skin Dendritic Cells.

Adenosine (Ado) produced by skin and skin migratory CD73+ dendritic cells is critically involved in tolerance to haptens. We therefore investigated the use of Ado receptor agonists for the treatment of contact hypersensitivity reactions. A2A- 4-[2-[[6-Amino-9-(N-ethyl-ß-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino] ethyl]benzenepropanoic acid hydrochloride (CGS) and A2B- 2-[[6-Amino-3,5-dicyano-4-[4-[cyclopropylmethoxy]phenyl]-2-pyridinyl]thio]-acetamide (BAY) specific Ado receptor agonists were epicutaneously applied to the skin before sensitization and challenge with DNFB. Both agonists reduced ear swelling compared with solvent controls. This was accompanied by fewer activated T cells in the skin after the challenge and by higher numbers of T cells expressing anergic markers such as LAG-3, CD137, PD-1, CD272, and TIM-3 in the lymph nodes of CGS-treated groups. In ear tissue, Ado receptor agonist treatment reduced the production of proinflammatory cytokines and chemokines as well as the infiltration by neutrophils after sensitization. Moreover, reduced numbers of skin migratory dendritic cells producing less IL-12 and exhibiting lower expression of CD86 were recorded in lymph nodes after sensitization. In cocultures of skin migratory dendritic cells from CGS-treated mice with T cells, reduced proliferation of T cells and decreased secretion of proinflammatory cytokines compared with that of solvent controls were apparent. In conclusion, topical application of Ado receptor agonists to the skin prevents sensitization of T cells against haptens by reducing the migration and activation of skin migratory dendritic cells.

Bullous pemphigoid (BP) patients with selective IgG autoreactivity against BP230: Review of a rare but valuable cohort with impact on the comprehension of the pathogenesis of BP.

Bullous pemphigoid (BP) is the most common autoimmune blistering disease. BP is characterized by the development of tense blisters induced by tissue-bound specific autoantibodies directed against the major autoantigens bullous pemphigoid autoantigen 180 (BP180, also called BPAG2 or Collagen XVII) and bullous pemphigoid autoantigen 230 (BP230, also called BPAG1 or dystonin). The vast majority of BP patients have autoantibodies targeting BP180, or both, BP180 and BP230. The hemidesmosomal protein BP180 is regarded as the main autoantigen, whereas the pathophysiologic relevance of intracellularly-located BP230 is controversial. A small subpopulation of BP patients selectively reveals autoantibodies against BP230 (BP230+ patients) strongly supporting that BP230 autoantibodies might be sufficient to induce skin pathology. In line, BP animal models have been developed, which successfully mimic a human BP phenotype by targeting BP230. In this context, our group has recently shown that a murine autoantibody targeting BP230 induces subepidermal blisters in vivo. This finding suggests that blister formation in the population of patients with selective autoreactivity against BP230 may share pathophysiologic features of pathogenic anti-BP230 autoantibodies in our murine model. This review summarizes the clinical features of BP patients with selective autoreactivity against BP230, enlightens the currently available BP mouse models targeting BP230 and discusses the potential pathophysiological mechanism of BP230 autoantibodies.

The Role of KH-Type Splicing Regulatory Protein (KSRP) for Immune Functions and Tumorigenesis.

Post-transcriptional control of gene expression is one important mechanism that enables stringent and rapid modulation of cytokine, chemokines or growth factors expression, all relevant for immune or tumor cell function and communication. The RNA-binding protein KH-type splicing regulatory protein (KSRP) controls the mRNA stability of according genes by initiation of mRNA decay and inhibition of translation, and by enhancing the maturation of microRNAs. Therefore, KSRP plays a pivotal role in immune cell function and tumor progression. In this review, we summarize the current knowledge about KSRP with regard to the regulation of immunologically relevant targets, and the functional role of KSRP on immune responses and tumorigenesis. KSRP is involved in the control of myeloid hematopoiesis. Further, KSRP-mediated mRNA decay of pro-inflammatory factors is necessary to keep immune homeostasis. In case of infection, functional impairment of KSRP is important for the induction of robust immune responses. In this regard, KSRP seems to primarily dampen T helper cell 2 immune responses. In cancer, KSRP has often been associated with tumor growth and metastasis. In summary, aside of initiation of mRNA decay, the KSRP-mediated regulation of microRNA maturation seems to be especially important for its diverse biological functions, which warrants further in-depth examination.

ß2 Integrins on Dendritic Cells Modulate Cytokine Signaling and Inflammation-Associated Gene Expression, and Are Required for Induction of Autoimmune Encephalomyelitis.

Heterodimeric ß2 integrin surface receptors (CD11a-d/CD18) are specifically expressed by leukocytes that contribute to pathogen uptake, cell migration, immunological synapse formation and cell signaling. In humans, the loss of CD18 expression results in leukocyte adhesion deficiency syndrome (LAD-)1, largely characterized by recurrent severe infections. All available mouse models display the constitutive and ubiquitous knockout of either α or the common ß2 (CD18) subunit, which hampers the analysis of the cell type-specific role of ß2 integrins in vivo. To overcome this limitation, we generated a CD18 gene floxed mouse strain. Offspring generated from crossing with CD11c-Cre mice displayed the efficient knockdown of ß2 integrins, specifically in dendritic cells (DCs). Stimulated ß2-integrin-deficient splenic DCs showed enhanced cytokine production and the concomitantly elevated activity of signal transducers and activators of transcription (STAT) 1, 3 and 5, as well as the impaired expression of suppressor of cytokine signaling (SOCS) 2-6 as assessed in bone marrow-derived (BM) DCs. Paradoxically, these BMDCs also showed the attenuated expression of genes involved in inflammatory signaling. In line, in experimental autoimmune encephalomyelitis mice with a conditional DC-specific ß2 integrin knockdown presented with a delayed onset and milder course of disease, associated with lower frequencies of T helper cell populations (Th)1/Th17 in the inflamed spinal cord. Altogether, our mouse model may prove to be a valuable tool to study the leukocyte-specific functions of ß2 integrins in vivo.