Pre-clinical proof-of-concept of anti-fibrotic activity of caveolin-1 scaffolding domain peptide LTI-03 in ex vivo precision cut lung slices from patients with Idiopathic Pulmonary Fibrosis.

Rationale: While rodent lung fibrosis models are routinely used to evaluate novel antifibrotics, these models have largely failed to predict clinical efficacy of novel drug candidates for Idiopathic Pulmonary Fibrosis (IPF). Moreover, single target therapeutic strategies for IPF have failed and current multi-target standard of care drugs are not curative. Caveolin-1 (CAV-1) is an integral membrane protein, which, via its caveolin scaffolding domain (CSD), interacts with caveolin binding domains (CBD). CAV-1 regulates homeostasis, and its expression is decreased in IPF lungs. LTI-03 is a seven amino acid peptide derived from the CSD and formulated for dry powder inhalation; it was well tolerated in normal volunteers ( NCT04233814 ) and a safety trial is underway in IPF patients ( NCT05954988 ). Objectives: Anti-fibrotic efficacy of LTI-03 and other CSD peptides has been observed in IPF lung monocultures, and rodent pulmonary, dermal, and heart fibrosis models. This study aimed to characterize progressive fibrotic activity in IPF PCLS explants and to evaluate the antifibrotic effects of LTI-03 and nintedanib in this model. Methods: First, CBD regions were identified in IPF signaling proteins using in silico analysis. Then, IPF PCLS (n=8) were characterized by COL1A1 immunostaining, multiplex immunoassays, and bulk RNA sequencing following treatment every 12hrs with LTI-03 at 0.5, 3.0, or 10 µM; nintedanib at 0.1 µM or 1 µM; or control peptide (CP) at 10 µM. Measurements and Main Results: CBDs were present in proteins implicated in IPF, including VEGFR, FGFR and PDGFR. Increased expression of profibrotic mediators indicated active fibrotic activity in IPF PCLS over five days. LTI-03 dose dependently decreased COL1A1 staining, and like nintedanib, decreased profibrotic proteins and transcripts. Unlike nintedanib, LTI-03 did not induce cellular necrosis signals. Conclusion: IPF PCLS explants demonstrate molecular activity indicative of fibrosis during 5 days in culture and LTI-03 broadly attenuated pro-fibrotic proteins and pathways, further supporting the potential therapeutic effectiveness of LTI-03 for IPF.

Caveolin scaffolding domain (CSD) peptide LTI-2355 modulates the phagocytic and synthetic activity of lung derived myeloid cells in Idiopathic Pulmonary Fibrosis (IPF) and Post-acute sequelae of COVID-fibrosis (PASC-F).

Rationale: The role of the innate immune system in Idiopathic Pulmonary Fibrosis (IPF) remains poorly understood. However, a functional myeloid compartment is required to remove dying cells and cellular debris, and to mediate innate immune responses against pathogens. Aberrant macrophage activity has been described in patients with Post-acute sequelae of COVID fibrosis (PASC-F). Therefore, we examined the functional and synthetic properties of myeloid cells isolated from normal donor lung and lung explant tissue from both IPF and PASC-F patients and explored the effect of LTI-2355, a Caveolin Scaffolding Domain (CSD) peptide, on these cells. Methods & Results: CD45 + myeloid cells isolated from lung explant tissue from IPF and PASC-F patients exhibited an impaired capacity to clear autologous dead cells and cellular debris. Uptake of pathogen-coated bioparticles was impaired in myeloid cells from both fibrotic patient groups independent of type of pathogen highlighting a cell intrinsic functional impairment. LTI-2355 improved the phagocytic activity of both IPF and PASC-F myeloid cells, and this improvement was paired with decreased pro-inflammatory and pro-fibrotic synthetic activity. LTI-2355 was also shown to primarily target CD206-expressing IPF and PASC-F myeloid cells. Conclusions: Primary myeloid cells from IPF and PASC-F patients exhibit dysfunctional phagocytic and synthetic properties that are reversed by LTI-2355. Thus, these studies highlight an additional mechanism of action of a CSD peptide in the treatment of IPF and progressive fibrotic lung disease.

NOD2 drives early IL-33-dependent expansion of group 2 innate lymphoid cells during Crohn's disease-like ileitis.

Innate lymphoid cells (ILCs) are enriched at barrier surfaces, including the gastrointestinal tract. While most studies have focused on the balance between pathogenic group 1 ILCs (ILC1s) and protective ILC3s in maintaining gut homeostasis and during chronic intestinal inflammation, such as Crohn's disease (CD), less is known regarding ILC2s. Using an established murine model of CD-like ileitis, i.e., the SAMP1/YitFc (SAMP) mouse strain, we showed that ILC2s, compared with ILC1s and ILC3s, were increased within draining mesenteric lymph nodes and ilea of SAMP versus AKR (parental control) mice early, during the onset of disease. Gut-derived ILC2s from CD patients versus healthy controls were also increased and expanded, similarly to ILC1s, in greater proportion compared with ILC3s. Importantly, we report that the intracellular bacteria-sensing protein, nucleotide-binding oligomerization domaining-containing protein 2, encoded by Nod2, the first and strongest susceptibility gene identified for CD, promoted ILC2 expansion, which was dramatically reduced in SAMP mice lacking NOD2 and in SAMP mice raised under germ-free conditions. Furthermore, these effects occurred through a mechanism involving the IL-33/ST2 ligand-receptor pair. Collectively, our results indicate a functional link between NOD2 and ILC2s, regulated by the IL-33/ST2 axis, that mechanistically may contribute to early events leading to CD pathogenesis.

Biological Therapy in Inflammatory Bowel Disease Patients Partly Restores Intestinal Innate Lymphoid Cell Subtype Equilibrium.

Patients with Crohn disease (CD) and ulcerative colitis (UC) suffer from chronic relapsing intestinal inflammation. While many studies focused on adaptive immunity, less is known about the role of innate immune cells in these diseases. Innate lymphoid cells (ILCs) are recently identified cells with a high cytokine-producing capacity at mucosal barriers. The aim was to study the impact of biological treatment on ILC in CD and UC. Patients initiating anti-tumor necrosis factor (TNF), ustekinumab, or vedolizumab treatment were prospectively followed up and peripheral and intestinal ILCs were determined. In the inflamed gut tissue of patients with inflammatory bowel disease, we found an increase of ILC1 and in immature NKp44- ILC3, whereas there was a decrease of mature NKp44+ ILC3 when compared to healthy controls (HCs). Similar but less pronounced changes in ILC1 were observed in blood, whereas circulating NKp44- ILC3 were decreased. Fifteen percent of CD patients had NKp44+ ILC3 in blood and these cells were not detected in blood of HCs or UC patients. Therapy with three different biologicals (ustekinumab targeting the IL-12/23 cytokines, anti-TNF and vedolizumab) partly restored intestinal ILC subset equilibrium with a decrease of ILC1 (except for ustekinumab) and an increase of NKp44+ ILC3. Anti-TNF also mobilized more NKp44+ ILC3 in circulation. As ILC1 are proinflammatory cells and as NKp44+ ILC3 contribute to homeostasis of intestinal mucosa, the observed effects of biologicals on ILCs might contribute to their clinical efficacy.

Fibrogenesis in chronic murine colitis is independent of innate lymphoid cells.

INTRODUCTION: Insight in the pathogenesis of intestinal fibrosis is an unmet medical need in inflammatory bowel diseases. Studies in murine models and human organ fibrosis point to a potential role of innate lymphoid cells (ILC) in chronic intestinal inflammation and fibrosis. MATERIALS AND METHODS: Dextran sodium sulfate (DSS) in drinking water was used to induce chronic colitis and remodeling in C57Bl/6 wild type (WT), RAG-deficient, RAG-/- common γ chain deficient and anti-CD90.2 monoclonal antibody treated RAG-/- mice. Inflammation was scored by macroscopic and histological examination and fibrosis was evaluated by hydroxyproline quantification and histology. RESULTS: In RAG-/- mice (which have a normal ILC population but no adaptive immunity), chronic intestinal inflammation and fibrosis developed similarly as in WT mice, with a relative increase in ILC2 during repeated DSS exposure. Chronic colitis could also be induced in the absence of ILC (RAG-/- γc-/- or anti-CD90.2 treated RAG-/- mice) with no attenuation of fibrosis. Importantly, clinical recovery based on weight gain after stopping DSS exposure was impaired in ILC-deficient or ILC-depleted mice. CONCLUSION: These data argue against a profibrotic effect of ILC in chronic colitis, but rather suggest that ILC have a protective and recovery-enhancing effect after repeated intestinal injury.

Fibrogenesis in Chronic DSS Colitis is Not Influenced by Neutralisation of Regulatory T Cells, of Major T Helper Cytokines or Absence of IL-13.

Mechanisms underlying fibrogenesis in chronic colitis are largely unknown. There is an urgent need for clinical markers and identification of targets to prevent, treat and limit intestinal fibrosis. This study investigated the contribution of major T cell cytokines and T regulatory cells (Tregs) to inflammation and fibrosis induced in a model of experimental colitis by oral intake of dextran sodium sulphate (DSS) in wild type and IL-13 knock-out C57Bl/6 mice. Inflammation and fibrosis were scored by macroscopic and histological examination and fibrosis was quantified by hydroxyproline. Numbers of Tregs and IFN-γ+, IL-13+ and IL-17A+ CD4+ T helper (Th) cells in mesenteric lymph nodes increased during chronic DSS administration and mRNA for IFN-γ and IL-17 in the inflamed colon tissue was upregulated. However, antibody-mediated neutralisation of IFN-γ or IL-17A/F in a therapeutic setting had no effect on chronic intestinal inflammation and fibrosis. Antibody-mediated depletion of Tregs did not enhance fibrosis, nor did IL-13 deficiency have an effect on the fibrotic disease. These data argue against an important contribution of Tregs and of the cytokines IFN-γ, IL-13, IL-17A, IL-17F in the induction and/or control of fibrosis in this Crohn's disease like murine model.

Mucosal IL13RA2 expression predicts nonresponse to anti-TNF therapy in Crohn's disease.

BACKGROUND: Ileocolonic expression of IL13RA2 has been identified as a predictive marker for nonresponsiveness to infliximab (IFX) in patients with Crohn's disease (CD). AIM: To validate the IL13RA2 biomarker, study its anti-TNF specificity and get a better understanding of the underlying biology driving its expression. METHODS: IL13RA2 mucosal expression was studied in a cohort of adalimumab and vedolizumab treated patients. To identify the upstream regulators of anti-TNF nonresponsiveness, weighted gene co-expression network analysis was applied on publicly available microarray data of IFX-treated patients. Selected serum proteins, including TNF, were measured prior to first IFX exposure and compared between healers and nonhealers. RESULTS: Increased mucosal IL13RA2 expression prior to start of biological therapy was predictive for anti-TNF nonresponsiveness specifically (AUROC, area under the curve = 0.90, P < 0.001 in anti-TNF vs AUROC = 0.63, P = 0.30 in vedolizumab treated patients). In baseline biopsies, TNF-driven pathways were significantly enriched in future anti-TNF nonhealers (P = 5.0 × 10-34 ). We found an increased baseline mucosal TNF burden in nonhealers (P = 0.02), and TNF mRNA correlated significantly with IL13RA2 expression (ρ = 0.55, P = 0.02). Baseline serum TNF levels were significantly lower in nonhealers (P = 0.04), and correlated inversely with IFX serum induction levels (r = -0.45, P = 0.002 at week 6). CONCLUSIONS: Increased mucosal IL13RA2 expression is associated with an increased mucosal TNF burden in CD patients. In view of its specificity for prediction of anti-TNF therapy resistance, mucosal IL13RA2 expression is a potential biomarker for therapy selection and/or for the need of increased anti-TNF drug dosing.

Effects of Epithelial IL-13Rα2 Expression in Inflammatory Bowel Disease.

Background: Mucosal IL-13 Receptor alpha 2 (IL13RA2) mRNA expression is one of the best predictive markers for primary non-responsiveness to infliximab therapy in patients with inflammatory bowel disease (IBD). The objective of this study was to understand how IL-13Rα2, a negative regulator of IL-13 signaling, can contribute to IBD pathology. Methods:IL13RA2 knockout (KO) and wild type (WT) mice were exposed to dextran sodium sulfate (DSS) in drinking water to induce colitis. Furthermore, mucosal biopsies and resection specimen of healthy individuals and IBD patients before the start of anti-tumor necrosis factor (anti-TNF) therapy were obtained for immunohistochemistry and gene expression analysis. Results: After induction of DSS colitis, IL13RA2 KO mice had similar disease severity, but recovered more rapidly than WT animals. Goblet cell numbers and mucosal architecture were also more rapidly restored in IL13RA2 KO mice. In mucosal biopsies of active IBD patients, immunohistochemistry revealed that IL-13Rα2 protein was highly expressed in epithelial cells, while expression was restricted to goblet cells in healthy controls. Mucosal IL13RA2 mRNA negatively correlated with mRNA of several goblet cell-specific and barrier genes, and with goblet cell numbers. Conclusions: The data suggest that IL-13Rα2 on epithelial cells contributes to IBD pathology by negatively influencing goblet cell recovery, goblet cell function and epithelial restoration after injury. Therefore, blocking IL-13Rα2 could be a promising target for restoration of the epithelial barrier in IBD.

Immunotherapy with subcutaneous immunogenic autologous tumor lysate increases murine glioblastoma survival.

Immunotherapeutic strategies for glioblastoma, the most frequent malignant primary brain tumor, aim to improve its disastrous consequences. On top of the standard treatment, one strategy uses T cell activation by autologous dendritic cells (DC) ex vivo loaded with tumor lysate to attack remaining cancer cells. Wondering whether 'targeting' in vivo DCs could replace these ex vivo ones, immunogenic autologous tumor lysate was used to treat glioma-inoculated mice in the absence of ex vivo loaded DCs. Potential immune mechanisms were studied in two orthotopic, immunocompetent murine glioma models. Pre-tumoral subcutaneous lysate treatment resulted in a survival benefit comparable to subcutaneous DC therapy. Focussing on the immune response, glioma T cell infiltration was observed in parallel with decreased amounts of regulatory T cells. Moreover, these results were accompanied by the presence of strong tumor-specific immunological memory, shown by complete survival of a second glioblastoma tumor, inoculated 100 days after the first one. Finally, in combination with temozolomide, survival of established glioma in mice could be increased. Our results show the potential of immunogenic autologous tumor lysate used to treat murine glioblastoma, which will be worthwhile to study in clinical trials as it has potential as a cost-efficient adjuvant treatment strategy for gliomas.