Fibrogenic Disorders in Human Diseases: From Inflammation to Organ Dysfunction.

Fibrosis is an inadequate response to tissue stress with very few therapeutic options to prevent its progression to organ dysfunction. There is an urgent need to identify drugs with a therapeutic potential for fibrosis, either by designing and developing new compounds or by repurposing drugs already in clinical use which were developed for other indications. In this Perspective, we summarize some pathways and biological targets involved in fibrosis development and maintenance, focusing on common mechanisms between organs and diseases. We review the therapeutic agents under experimental development, clinical trials, or in clinical use for the treatment of fibrotic disorders, evaluating the reasons for the discrepancies observed between preclinical and clinical results. We also discuss the improvement that we envision in the development of therapeutic molecules able to achieve improved potential for treatment, including indirect modulators, targeting approaches, or drug combinations

IL-2-Mediated In Vivo Expansion of Regulatory T Cells Combined with CD154-CD40 Co-Stimulation Blockade but Not CTLA-4 Ig Prolongs Allograft Survival in Naive and Sensitized Mice.

In recent years, regulatory T cells (Treg)-based immunotherapy has emerged as a promising strategy to promote operational tolerance after solid organ transplantation (SOT). However, a main hurdle for the therapeutic use of Treg in transplantation is their low frequency, particularly in non-lymphopenic hosts. We aimed to expand Treg directly in vivo and determine their efficacy in promoting donor-specific tolerance, using a stringent experimental model. Administration of the IL-2/JES6-1 immune complex at the time of transplantation resulted in significant expansion of donor-specific Treg, which suppressed alloreactive T cells. IL-2-mediated Treg expansion in combination with short-term CD154-CD40 co-stimulation blockade, but not CTLA-4 Ig or rapamycin, led to tolerance to MHC-mismatched skin grafts in non-lymphopenic mice, mainly by hindering alloreactive CD8(+) effector T cells and the production of alloantibodies. Importantly, this treatment also allowed prolonged survival of allografts in the presence of either donor-specific or cross-reactive memory cells. However, late rejection occurred in sensitized hosts, partly mediated by activated B cells. Overall, these data illustrate the potential but also some important limitations of Treg-based therapy in clinical SOT as well as the importance of concomitant immunomodulatory strategies in particular in sensitized hosts

Fibroblast activation protein-α in fibrogenic disorders and cancer: more than a prolyl-specific peptidase?

Fibroblast activation protein-α (FAP-α) belongs to the family of prolyl-specific serine proteases. FAP-α displays both exopeptidase and endopeptidase/gelatinase/collagenase activities. FAP-α protein and/or activity have been associated with fibrosis, inflammation and cancer, but the protein is undetectable in most normal tissues. FAP-α is selectively expressed at sites of tissue remodeling and repair and enhances tumor progression, suggesting that this protease may be a therapeutic target to treat human disorders associated with fibrotic dysregulation. Areas covered: In this review, we summarize the mechanisms driving tissue fibrosis and describe some of the enzymes involved in fibrosis, concentrating on FAP-α. We describe its enzymatic properties, discuss the tools developed to control its activity and the problem of selectivity toward the other proteases of the family and outline its potential biological substrates. We also consider non-enzymatic functions of this protein and suggest that repression of FAP-α expression may represent therapeutic options. Expert opinion: Questions remain regarding the biological functions of FAP-α, either dependent or independent of its enzyme activity. However, as progress is underway to develop FAP-α-specific inhibitors and therapeutic antibodies, its role in diseases associated with fibrosis is starting to emerge, ultimately leading to novel therapeutic options for inflammatory and oncologic diseases

Myeloid-derived suppressor cells are implicated in regulating permissiveness for tumor metastasis during mouse gestation.

Metastasis depends on the ability of tumor cells to establish a relationship with the newly seeded tissue that is conducive to their survival and proliferation. However, the factors that render tissues permissive for metastatic tumor growth have yet to be fully elucidated. Breast tumors arising during pregnancy display early metastatic proclivity, raising the possibility that pregnancy may constitute a physiological condition of permissiveness for tumor dissemination. Here we have shown that during murine gestation, metastasis is enhanced regardless of tumor type, and that decreased NK cell activity is responsible for the observed increase in experimental metastasis. Gene expression changes in pregnant mouse lung and liver were shown to be similar to those detected in premetastatic sites and indicative of myeloid cell infiltration. Indeed, myeloid-derived suppressor cells (MDSCs) accumulated in pregnant mice and exerted an inhibitory effect on NK cell activity, providing a candidate mechanism for the enhanced metastatic tumor growth observed in gestant mice. Although the functions of MDSCs are not yet understood in the context of pregnancy, our observations suggest that they may represent a shared mechanism of immune suppression occurring during gestation and tumor growth

Differential Effects of the Mitochondria-Active Tetrapeptide SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH₂) and Its Peptidase-Targeted Prodrugs in Experimental Acute Kidney Injury.

The mitochondria-active tetrapeptide SS-31 can control oxidative tissue damage in kidney diseases. To investigate other potential beneficial nephroprotective effects of SS-31, in vivo murine models of acute tubular injury and glomerular damage were developed. Reduction of acute kidney injury was demonstrated in mice treated with SS-31. The expression of mRNAs involved in acute inflammatory and oxidative stress responses in the diseased kidneys confirmed that SS-31 could regulate these pathways in our in vivo models. Furthermore, ex vivo histoenzymography of mouse kidneys showed that aminopeptidase A (APA), the enzyme involved in the processing of angiotensin (Ang) II to Ang III, was induced in the diseased kidneys, and its activity was inhibited by SS-31. As the renin-angiotensin system (RAS) is a main regulator of kidney functions, the modulation of Ang receptors (ATR) and APA by SS-31 was further investigated using mRNAs extracted from diseased kidneys. Following acute tubular and/or glomerular damage, the expression of the AT <sub>1</sub> R mRNA was upregulated, which could be selectively downregulated upon SS-31 administration to the animals. At the same time, SS-31 was able to increase the expression of the AT <sub>2</sub> R, which may contribute to limit renal damage. Consequently, SS-31-based prodrugs were developed as substrates and/or inhibitors for APA and were screened using cells expressing high levels of APA, showing its selective regulation by α-Glu-SS-31. Thus, a link between SS-31 and the RAS opens new therapeutic implications for SS-31 in kidney diseases

Lipoxin A₄ prevents the progression of de novo and established endometriosis in a mouse model by attenuating prostaglandin E₂ production and estrogen signaling.

Endometriosis, a leading cause of pelvic pain and infertility, is characterized by ectopic growth of endometrial-like tissue and affects approximately 176 million women worldwide. The pathophysiology involves inflammatory and angiogenic mediators as well as estrogen-mediated signaling and novel, improved therapeutics targeting these pathways are necessary. The aim of this study was to investigate mechanisms leading to the establishment and progression of endometriosis as well as the effect of local treatment with Lipoxin A4 (LXA₄), an anti-inflammatory and pro-resolving lipid mediator that we have recently characterized as an estrogen receptor agonist. LXA₄ treatment significantly reduced endometriotic lesion size and downregulated the pro-inflammatory cytokines IL-1β and IL-6, as well as the angiogenic factor VEGF. LXA₄ also inhibited COX-2 expression in both endometriotic lesions and peritoneal fluid cells, resulting in attenuated peritoneal fluid Prostaglandin E₂ (PGE₂) levels. Besides its anti-inflammatory effects, LXA₄ differentially regulated the expression and activity of the matrix remodeling enzyme matrix metalloproteinase (MMP)-9 as well as modulating transforming growth factor (TGF)-β isoform expression within endometriotic lesions and in peritoneal fluid cells. We also report for first time that LXA₄ attenuated aromatase expression, estrogen signaling and estrogen-regulated genes implicated in cellular proliferation in a mouse model of disease. These effects were observed both when LXA₄ was administered prior to disease induction and during established disease. Collectively, our findings highlight potential targets for the treatment of endometriosis and suggest a pleotropic effect of LXA₄ on disease progression, by attenuating pro-inflammatory and angiogenic mediators, matrix remodeling enzymes, estrogen metabolism and signaling, as well as downstream proliferative pathways

Treg-Therapy Allows Mixed Chimerism and Transplantation Tolerance Without Cytoreductive Conditioning

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short-course costimulation blockade and rapamycin. This combination treatment led to long-term multilineage chimerism and donor-specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3-transduced Tregs, natural Tregs and TGF-β induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach

Mutually exclusive lymphangiogenesis or perineural infiltration in human skin squamous-cell carcinoma.

Although tumor-associated lymphangiogenesis correlates with metastasis and poor prognosis in several cancers, it also supports T cell infiltration into the tumor and predicts favorable outcome to immunotherapy. The role of lymphatic vessels in skin squamous-cell carcinoma (sSCC), the second most common form of skin cancer, remains mostly unknown. Although anti-PD-1 therapy is beneficial for some patients with advanced sSCC, a greater understanding of disease mechanisms is still needed to develop better therapies. Using quantitative multiplex immunohistochemistry, we analyzed sSCC sections from 36 patients. CD8+ T cell infiltration showed great differences between patients, whereby these cells were mainly excluded from the tumor mass. Similar to our data in melanoma, sSCC with high density of lymphatic endothelial cells showed increased CD8+ T cell density in tumor areas. An entirely new observation is that sSCC with perineural infiltration but without metastasis was characterized by low lymphatic endothelial cell density. Since both, metastasis and perineural infiltration are known to affect tumor progression and patients' prognosis, it is important to identify the molecular drivers, opening future options for therapeutic targeting. Our data suggest that the mechanisms underlying perineural infiltration may be linked with the biology of lymphatic vessels and thus stroma

SARS-CoV-2 / COVID-19 in patients on the Swiss national transplant waiting list.

The impact of coronavirus disease 2019 (COVID-19) on patients listed for solid organ transplantation has not been systematically investigated to date. Thus, we assessed occurrence and effects of infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on patients on the Swiss national waiting list for solid organ transplantation. Patient data were retrospectively extracted from the Swiss Organ Allocation System (SOAS). From 16 March to 31 May 2020, we included all patients listed for solid organ transplantation on the Swiss national waiting list who were tested positive for SARS-CoV-2. Severity of COVID-19 was categorised as follows: stage I, mild symptoms; stage II, moderate to severe symptoms; stage III, critical symptoms; stage IV, death. We compared the incidence rate (laboratory-confirmed cases of SARS-CoV-2), the hospital admission rate (number of admissions of SARS-CoV-2-positive individuals), and the case fatality rate (number of deaths of SARS-CoV-2-positive individuals) in our study population with the general Swiss population during the study period, calculating age-adjusted standardised incidence ratios and standardised mortality ratios, with 95% confidence intervals (CIs). A total of 1439 patients were registered on the Swiss national solid organ transplantation waiting list on 31 May 31 2020. Twenty-four (1.7%) waiting list patients were reported to test positive for SARS-CoV-2 in the study period. The median age was 56 years (interquartile range 45.3–65.8), and 14 (58%) were male. Of all patients tested positive for SARS-CoV-2, two patients were asymptomatic, 14 (58%) presented in COVID-19 stage I, 3 (13%) in stage II, and 5 (21%) in stage III. Eight patients (33%) were admitted to hospital, four (17%) required intensive care, and three (13%) mechanical ventilation. Twenty-two patients (92%) of all those infected recovered, but two male patients aged >65 years with multiple comorbidities died in hospital from respiratory failure. Comparing our study population with the general Swiss population, the age-adjusted standardised incidence ratio was 4.1 (95% CI 2.7–6.0). The overall rate of SARS-CoV-2 infections in candidates awaiting solid organ transplantation was four times higher than in the Swiss general population; however, the frequency of testing likely played a role. Given the small sample size of affected patients, conclusions have to be drawn cautiously and results need verification in larger cohorts

Targeted γ-secretase inhibition of Notch signaling activation in acute renal injury.

The Notch pathway has been reported to control tissue damage in acute kidney diseases. To investigate potential beneficial nephroprotective effects of targeting Notch, we developed chemically functionalized γ-secretase inhibitors (GSIs) targeting γ-glutamyltranspeptidase (γ-GT) and/or γ-glutamylcyclotransferase (γ-GCT), two enzymes overexpressed in the injured kidney, and evaluated them in in vivo murine models of acute tubular and glomerular damage. Exposure of the animals to disease-inducing drugs together with the functionalized GSIs improved proteinuria and, to some extent, kidney dysfunction. The expression of genes involved in the Notch pathway, acute inflammatory stress responses, and the renin-angiotensin system was enhanced in injured kidneys, which could be downregulated upon administration of functionalized GSIs. Immunohistochemistry staining and Western blots demonstrated enhanced activation of Notch1 as detected by its cleaved active intracellular domain during acute kidney injury, and this was downregulated by concomitant treatment with the functionalized GSIs. Thus targeted γ-secretase-based prodrugs developed as substrates for γ-GT/γ-GCT have the potential to selectively control Notch activation in kidney diseases with subsequent regulation of the inflammatory stress response and the renin-angiotensin pathways