Shared and Distinct Renal Transcriptome Signatures in 3 Standard Mouse Models of Chronic Kidney Disease.

INTRODUCTION: Several mouse models with diverse disease etiologies are used in preclinical research for chronic kidney disease (CKD). Here, we performed a head-to-head comparison of renal transcriptome signatures in standard mouse models of CKD to assess shared and distinct molecular changes in three mouse models commonly employed in preclinical CKD research and drug discovery. METHODS: All experiments were conducted on male C57BL/6J mice. Mice underwent sham, unilateral ureter obstruction (UUO), or unilateral ischemic-reperfusion injury (uIRI) surgery and were terminated two- and 6-weeks post-surgery, respectively. The adenine-supplemented diet-induced (ADI) model of CKD was established by feeding with adenine diet for 6 weeks and compared to control diet feeding. For all models, endpoints included plasma biochemistry, kidney histology, and RNA sequencing. RESULTS: All models displayed increased macrophage infiltration (F4/80 IHC) and fibrosis (collagen 1a1 IHC). Compared to corresponding controls, all models were characterized by an extensive number of renal differentially expressed genes (≥11,000), with a notable overlap in transcriptomic signatures across models. Gene expression markers of fibrosis, inflammation, and kidney injury supported histological findings. Interestingly, model-specific transcriptome signatures included several genes representing current drug targets for CKD, emphasizing advantages and limitations of the three CKD models in preclinical target and drug discovery. CONCLUSION: The UUO, uIRI, and ADI mouse models of CKD have significant commonalities in their renal global transcriptome profile. Model-specific renal transcriptional signatures should be considered when selecting the specific model in preclinical target and drug discovery.

The proteolytic airway environment associated with pneumonia acts as a barrier for treatment with anti-infective antibodies.

Like pneumonia, coronavirus disease 2019 (COVID-19) is characterized by a massive infiltration of innate immune cells (such as polymorphonuclear leukocytes) into the airways and alveolar spaces. These cells release proteases that may degrade therapeutic antibodies and thus limit their effectiveness. Here, we investigated the in vitro and ex vivo impact on anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) IgG1s and other IgG subclasses (IgG2 and IgG4) of the neutrophil elastase, proteinase 3 and cathepsin G (the three main neutrophil serine proteases) found in endotracheal aspirates from patients with severe COVID-19. Although the IgGs were sensitive to neutrophil serine proteases, IgG2 was most resistant to proteolytic degradation. The two anti-SARS CoV2 antibodies (casirivimab and imdevimab) were sensitive to the lung's proteolytic environment, although neutrophil serine protease inhibitors only partly limited the degradation. Overall, our results show that the pneumonia-associated imbalance between proteases and their inhibitors in the airways contributes to degradation of antiviral antibodies.

Barriers for orally inhaled therapeutic antibodies.

INTRODUCTION: Respiratory diseases represent a worldwide health issue. The recent Sars-CoV-2 pandemic, the burden of lung cancer, and inflammatory respiratory diseases urged the development of innovative therapeutic solutions. In this context, therapeutic antibodies (Abs) offer a tremendous opportunity to benefit patients with respiratory diseases. Delivering Ab through the airways has been demonstrated to be relevant to improve their therapeutic index. However, few inhaled Abs are on the market. AREAS COVERED: This review describes the different barriers that may alter the fate of inhaled therapeutic Abs in the lungs at steady state. It addresses both physical and biological barriers and discusses the importance of taking into consideration the pathological changes occurring during respiratory disease, which may reinforce these barriers. EXPERT OPINION: The pulmonary route remains rare for delivering therapeutic Abs, with few approved inhaled molecules, despite promising evidence. Efforts must focus on the intertwined barriers associated with lung diseases to develop appropriate Ab-formulation-device combo, ensuring optimal Ab deposition in the respiratory tract. Finally, randomized controlled clinical trials should be carried out to establish inhaled Ab therapy as prominent against respiratory diseases.

Inhalable Nanofitin demonstrates high neutralization of SARS-CoV-2 virus via direct application in respiratory tract.

Nanofitins are small and hyperthermostable alternative protein scaffolds that display physicochemical properties making them suitable for the development of topical therapeutics, notably for the treatment of pulmonary infectious diseases. Local administration of biologics to the lungs involves a particularly stressful step of nebulization that is poorly tolerated by most antibodies, which limits their application by this delivery route. During the COVID-19 pandemic, we generated anti-SARS-CoV-2 monomeric Nanofitins of high specificity for the spike protein. Hit Nanofitin candidates were identified based on their binding properties with punctual spike mutants and assembled into a linear multimeric construction constituting of four different Nanofitins, allowing the generation of a highly potent anti-SARS-CoV-2 molecule. The therapeutic efficacy of the multimeric assembly was demonstrated both in in vitro and in vivo models. Interestingly, the neutralization mechanism of the multimeric construction seems to involve a particular conformation switch of the spike trimer. In addition, we reported the stability and the conserved activity of the tetrameric construction after nebulization. This advantageous developability feature for pulmonary administration associated with the ease of assembly, as well as the fast generation process position the Nanofitin technology as a potential therapeutic solution for emerging infectious diseases.

Mucosal administration of anti-bacterial antibodies provide long-term cross-protection against Pseudomonas aeruginosa respiratory infection.

Bacterial respiratory infections, either acute or chronic, are major threats to human health. Direct mucosal administration, through the airways, of therapeutic antibodies (Abs) offers a tremendous opportunity to benefit patients with respiratory infections. The mode of action of anti-infective Abs relies on pathogen neutralization and crystallizable fragment (Fc)-mediated recruitment of immune effectors to facilitate their elimination. Using a mouse model of acute pneumonia induced by Pseudomonas aeruginosa, we depicted the immunomodulatory mode of action of a neutralizing anti-bacterial Abs. Beyond the rapid and efficient containment of the primary infection, the Abs delivered through the airways harnessed genuine innate and adaptive immune responses to provide long-term protection, preventing secondary bacterial infection. In vitro antigen-presenting cells stimulation assay, as well as in vivo bacterial challenges and serum transfer experiments indicate an essential contribution of immune complexes with the Abs and pathogen in the induction of the sustained and protective anti-bacterial humoral response. Interestingly, the long-lasting response protected partially against secondary infections with heterologous P. aeruginosa strains. Overall, our findings suggest that Abs delivered mucosally promotes bacteria neutralization and provides protection against secondary infection. This opens novel perspectives for the development of anti-infective Abs delivered to the lung mucosa, to treat respiratory infections.

A Protective Role of NOD2 on Oxazolone-induced Intestinal Inflammation Through IL-1ß-mediated Signalling Pathway.

BACKGROUND AND AIMS: NOD2 has emerged as a critical player in the induction of both Th1 and Th2 responses for potentiation and polarisation of antigen-dependent immunity. Loss-of-function mutations in the NOD2-encoding gene and deregulation of its downstream signalling pathway have been linked to Crohn's disease. Although it is well documented that NOD2 is capable of sensing bacterial muramyl dipeptide, it remains counter-intuitive to link development of overt intestinal inflammation to a loss of bacterial-induced inflammatory response. We hypothesised that a T helper bias could also contribute to an autoimmune-like colitis different from inflammation that is fully fledged by Th1 type cells. METHODS: An oedematous bowel wall with a mixed Th1/Th2 response was induced in mice by intrarectal instillation of the haptenating agent oxazolone. Survival and clinical scoring were evaluated. At several time points after instillation, colonic damage was assessed by macroscopic and microscopic observations. To evaluate the involvement of NOD2 in immunochemical phenomena, quantitative polymerase chain reaction [PCR] and flow cytometry analysis were performed. Bone marrow chimera experimentation allowed us to evaluate the role of haematopoietic/non-hematopoietic NOD2-expressing cells. RESULTS: Herein, we identified a key regulatory circuit whereby NOD2-mediated sensing of a muramyl dipeptide [MDP] by radio-resistant cells improves colitis with a mixed Th1/Th2 response that is induced by oxazolone. Genetic ablation of either Nod2 or Ripk2 precipitated oxazolone colitis that is predominantly linked to a lack of interferon-gamma. Bone marrow chimera experiments revealed that inactivation of Nod2 signalling in non-haematopoietic cells is causing a biased M1-M2 polarisation of macrophages and a decreased frequency of splenic regulatory T cells that correlates with an impaired activation of CD4 + T cells within mesenteric lymph nodes. Mechanistically, mice were protected from oxazolone-induced colitis upon administration of MDP in an interleukin-1- and interleukin-23-dependent manner. CONCLUSIONS: These findings indicate that Nod2 signalling may prevent pathological conversion of T helper cells for maintenance of tissue homeostasis.

Alternative Routes of Administration for Therapeutic Antibodies-State of the Art.

BACKGROUND: For the past two decades, there has been a huge expansion in the development of therapeutic antibodies, with 6 to 10 novel entities approved each year. Around 70% of these Abs are delivered through IV injection, a mode of administration allowing rapid and systemic delivery of the drug. However, according to the evidence presented in the literature, beyond the reduction of invasiveness, a better efficacy can be achieved with local delivery. Consequently, efforts have been made toward the development of innovative methods of administration, and in the formulation and engineering of novel Abs to improve their therapeutic index. OBJECTIVE: This review presents an overview of the routes of administration used to deliver Abs, different from the IV route, whether approved or in the clinical evaluation stage. We provide a description of the physical and biological fundamentals for each route of administration, highlighting their relevance with examples of clinically-relevant Abs, and discussing their strengths and limitations. METHODS: We reviewed and analyzed the current literature, published as of the 1 April 2022 using MEDLINE and EMBASE databases, as well as the FDA and EMA websites. Ongoing trials were identified using clinicaltrials.gov. Publications and data were identified using a list of general keywords. CONCLUSIONS: Apart from the most commonly used IV route, topical delivery of Abs has shown clinical successes, improving drug bioavailability and efficacy while reducing side-effects. However, additional research is necessary to understand the consequences of biological barriers associated with local delivery for Ab partitioning, in order to optimize delivery methods and devices, and to adapt Ab formulation to local delivery. Novel modes of administration for Abs might in fine allow a better support to patients, especially in the context of chronic diseases, as well as a reduction of the treatment cost.

Nephroprotective Effects of Semaglutide as Mono- and Combination Treatment with Lisinopril in a Mouse Model of Hypertension-Accelerated Diabetic Kidney Disease.

Background: Obesity, hyperglycemia and hypertension are critical risk factors for development of diabetic kidney disease (DKD). Emerging evidence suggests that glucagon-like peptide-1 receptor (GLP-1R) agonists improve cardiovascular and renal outcomes in type 2 diabetes patients. Here, we characterized the effect of the long-acting GLP-1R agonist semaglutide alone and in combination with an ACE inhibitor (lisinopril) in a model of hypertension-accelerated, advanced DKD facilitated by adeno-associated virus-mediated renin overexpression (ReninAAV) in uninephrectomized (UNx) female diabetic db/db mice. Methods: Female db/db mice received a single intravenous injection of ReninAAV 1 week prior to UNx. Six weeks post-nephrectomy, db/db UNx-ReninAAV mice were administered (q.d.) vehicle, semaglutide (30 nmol/kg, s.c.) or semaglutide (30 nmol/kg, s.c.) + lisinopril (30 mg/kg, p.o.) for 11 weeks. Endpoints included blood pressure, plasma/urine biochemistry, kidney histopathology and RNA sequencing. Results: Vehicle-dosed db/db UNx-ReninAAV mice developed hallmarks of DKD characterized by severe albuminuria and advanced glomerulosclerosis. Semaglutide robustly reduced hyperglycemia, hypertension and albuminuria concurrent with notable improvements in glomerulosclerosis severity, podocyte filtration slit density, urine/renal kidney injury molecule-1 (KIM-1) levels and gene expression markers of inflammation and fibrogenesis in db/db UNx-ReninAAV mice. Co-administration of lisinopril further ameliorated hypertension and glomerulosclerosis. Conclusions: Semaglutide improves disease hallmarks in the db/db UNx-ReninAAV mouse model of advanced DKD. Further benefits on renal outcomes were obtained by adjunctive antihypertensive standard of care. Collectively, our study supports the development of semaglutide for management of DKD.

Aggregates Associated with Instability of Antibodies during Aerosolization Induce Adverse Immunological Effects.

BACKGROUND: Immunogenicity refers to the inherent ability of a molecule to stimulate an immune response. Aggregates are one of the major risk factors for the undesired immunogenicity of therapeutic antibodies (Ab) and may ultimately result in immune-mediated adverse effects. For Ab delivered by inhalation, it is necessary to consider the interaction between aggregates resulting from the instability of the Ab during aerosolization and the lung mucosa. The aim of this study was to determine the impact of aggregates produced during aerosolization of therapeutic Ab on the immune system. METHODS: Human and murine immunoglobulin G (IgG) were aerosolized using a clinically-relevant nebulizer and their immunogenic potency was assessed, both in vitro using a standard human monocyte-derived dendritic cell (MoDC) reporter assay and in vivo in immune cells in the airway compartment, lung parenchyma and spleen of healthy C57BL/6 mice after pulmonary administration. RESULTS: IgG aggregates, produced during nebulization, induced a dose-dependent activation of MoDC characterized by the enhanced production of cytokines and expression of co-stimulatory markers. Interestingly, in vivo administration of high amounts of nebulization-mediated IgG aggregates resulted in a profound and sustained local and systemic depletion of immune cells, which was attributable to cell death. This cytotoxic effect was observed when nebulized IgG was administered locally in the airways as compared to a systemic administration but was mitigated by improving IgG stability during nebulization, through the addition of polysorbates to the formulation. CONCLUSION: Although inhalation delivery represents an attractive alternative route for delivering Ab to treat respiratory infections, our findings indicate that it is critical to prevent IgG aggregation during the nebulization process to avoid pro-inflammatory and cytotoxic effects. The optimization of Ab formulation can mitigate adverse effects induced by nebulization.

Therapeutic antibodies - natural and pathological barriers and strategies to overcome them.

Antibody-based therapeutics have become a major class of therapeutics with over 120 recombinant antibodies approved or under review in the EU or US. This therapeutic class has experienced a remarkable expansion with an expected acceleration in 2021-2022 due to the extraordinary global response to SARS-CoV2 pandemic and the public disclosure of over a hundred anti-SARS-CoV2 antibodies. Mainly delivered intravenously, alternative delivery routes have emerged to improve antibody therapeutic index and patient comfort. A major hurdle for antibody delivery and efficacy as well as the development of alternative administration routes, is to understand the different natural and pathological barriers that antibodies face as soon as they enter the body up to the moment they bind to their target antigen. In this review, we discuss the well-known and more under-investigated extracellular and cellular barriers faced by antibodies. We also discuss some of the strategies developed in the recent years to overcome these barriers and increase antibody delivery to its site of action. A better understanding of the biological barriers that antibodies have to face will allow the optimization of antibody delivery near its target. This opens the way to the development of improved therapy with less systemic side effects and increased patients' adherence to the treatment.

Oral supplementation with yeast ß-glucans improves the resolution of Escherichia coli-associated inflammatory responses independently of monocyte/macrophage immune training.

Introduction: Confronted with the emerging threat of antimicrobial resistance, the development of alternative strategies to limit the use of antibiotics or potentiate their effect through synergy with the immune system is urgently needed. Many natural or synthetic biological response modifiers have been investigated in this context. Among them, ß-glucans, a type of soluble or insoluble polysaccharide composed of a linear or branched string of glucose molecules produced by various cereals, bacteria, algae, and inferior (yeast) and superior fungi (mushrooms) have garnered interest in the scientific community, with not less than 10,000 publications over the last two decades. Various biological activities of ß-glucans have been reported, such as anticancer, antidiabetic and immune-modulating effects. In vitro, yeast ß-glucans are known to markedly increase cytokine secretion of monocytes/macrophages during a secondary challenge, a phenomenon called immune training. Methods: Here, we orally delivered ß-glucans derived from the yeast S. cerevisiae to mice that were further challenged with Escherichia coli. Results: ß-glucan supplementation protected the mice from E. coli intraperitoneal and intra-mammary infections, as shown by a lower bacterial burden and greatly diminished tissue damage. Surprisingly, this was not associated with an increased local immune response. In addition, granulocyte recruitment was transient and limited, as well as local cytokine secretion, arguing for faster resolution of the inflammatory response. Furthermore, ex-vivo evaluation of monocytes/macrophages isolated or differentiated from ß-glucan-supplemented mice showed these cells to lack a trained response versus those from control mice. Conclusion: In conclusion, dietary ß-glucans can improve the outcome of Escherichia coli infections and dampen tissue damages associated to excessive inflammatory response. The mechanisms associated with such protection are not necessarily linked to immune system hyper-activation or immune training.

Integrative transcriptomic profiling of a mouse model of hypertension-accelerated diabetic kidney disease.

The current understanding of molecular mechanisms driving diabetic kidney disease (DKD) is limited, partly due to the complex structure of the kidney. To identify genes and signalling pathways involved in the progression of DKD, we compared kidney cortical versus glomerular transcriptome profiles in uninephrectomized (UNx) db/db mouse models of early-stage (UNx only) and advanced [UNxplus adeno-associated virus-mediated renin-1 overexpression (UNx-Renin)] DKD using RNAseq. Compared to normoglycemic db/m mice, db/db UNx and db/db UNx-Renin mice showed marked changes in their kidney cortical and glomerular gene expression profiles. UNx-Renin mice displayed more marked perturbations in gene components associated with the activation of the immune system and enhanced extracellular matrix remodelling, supporting histological hallmarks of progressive DKD in this model. Single-nucleus RNAseq enabled the linking of transcriptome profiles to specific kidney cell types. In conclusion, integration of RNAseq at the cortical, glomerular and single-nucleus level provides an enhanced resolution of molecular signalling pathways associated with disease progression in preclinical models of DKD, and may thus be advantageous for identifying novel therapeutic targets in DKD.

Immune Checkpoint and Anti-Angiogenic Antibodies for the Treatment of Non-Small Cell Lung Cancer in the European Union and United States.

Several types of antibodies (Abs) are currently used in non-small cell lung cancer (NSCLC). Anti-angiogenic and immune checkpoint inhibitor (ICI) Abs are the most frequent treatments used alone or with chemotherapy in metastatic NSCLC, for the front line and beyond. Considering the many therapeutic options for locally advanced and metastatic lung cancer and differences in use according to geographic area, we present here a comprehensive review of the marketed ICI and anti-angiogenic Abs approved in the European Union (EU) and the US to treat locally advanced and metastatic NSCLC patients. We briefly describe the different molecules and their development in thoracic oncology and compare pharmacokinetic data, processing decision algorithms and marketing authorizations by the EMA and US Food and Drug Administration (FDA).

Therapeutic effects of lisinopril and empagliflozin in a mouse model of hypertension-accelerated diabetic kidney disease.

Hypertension is a critical comorbidity for progression of diabetic kidney disease (DKD). To facilitate the development of novel therapeutic interventions with the potential to control disease progression, there is a need to establish translational animal models that predict treatment effects in human DKD. The present study aimed to characterize renal disease and outcomes of standard of medical care in a model of advanced DKD facilitated by adeno-associated virus (AAV)-mediated renin overexpression in uninephrectomized (UNx) db/db mice. Five weeks after single AAV administration and 4 wk after UNx, female db/db UNx-ReninAAV mice received (PO, QD) vehicle, lisinopril (40 mg/kg), empagliflozin (20 mg/kg), or combination treatment for 12 wk (n = 17 mice/group). Untreated db/+ mice (n = 8) and vehicle-dosed db/db UNx-LacZAAV mice (n = 17) served as controls. End points included plasma, urine, and histomorphometric markers of kidney disease. Total glomerular numbers and individual glomerular volume were evaluated by whole kidney three-dimensional imaging analysis. db/db UNx-ReninAAV mice developed hallmarks of progressive DKD characterized by severe albuminuria, advanced glomerulosclerosis, and glomerular hypertrophy. Lisinopril significantly improved albuminuria, glomerulosclerosis, tubulointerstitial injury, and inflammation. Although empagliflozin alone had no therapeutic effect on renal endpoints, lisinopril and empagliflozin exerted synergistic effects on renal histological outcomes. In conclusion, the db/db UNx-ReninAAV mouse demonstrates good clinical translatability with respect to physiological and histological hallmarks of progressive DKD. The efficacy of standard of care to control hypertension and hyperglycemia provides a proof of concept for testing novel drug therapies in the model.NEW & NOTEWORTHY Translational animal models of diabetic kidney disease (DKD) are important tools in preclinical research and drug discovery. Here, we show that the standard of care to control hypertension (lisinopril) and hyperglycemia (empagliflozin) improves physiological and histopathological hallmarks of kidney disease in a mouse model of hypertension-accelerated progressive DKD. The findings substantiate hypertension and type 2 diabetes as essential factors in driving DKD progression and provide a proof of concept for probing novel drugs for potential nephroprotective efficacy in this model.

Chronic Pseudomonas aeruginosa Lung Infection Is IL-1R Independent, but Relies on MyD88 Signaling.

Cystic fibrosis is associated with chronic Pseudomonas aeruginosa colonization and inflammation. The role of MyD88, the shared adapter protein of the proinflammatory TLR and IL-1R families, in chronic P. aeruginosa biofilm lung infection is unknown. We report that chronic lung infection with the clinical P. aeruginosa RP73 strain is associated with uncontrolled lung infection in complete MyD88-deficient mice with epithelial damage, inflammation, and rapid death. Then, we investigated whether alveolar or myeloid cells contribute to heightened sensitivity to infection. Using cell-specific, MyD88-deficient mice, we uncover that the MyD88 pathway in myeloid or alveolar epithelial cells is dispensable, suggesting that other cell types may control the high sensitivity of MyD88-deficient mice. By contrast, IL-1R1-deficient mice control chronic P. aeruginosa RP73 infection and IL-1ß Ab blockade did not reduce host resistance. Therefore, the IL-1R1/MyD88 pathway is not involved, but other IL-1R or TLR family members need to be investigated. Our data strongly suggest that IL-1 targeted neutralizing therapies used to treat inflammatory diseases in patients unlikely reduce host resistance to chronic P. aeruginosa infection.

Therapeutic Antibodies for the Treatment of Respiratory Tract Infections-Current Overview and Perspectives.

Respiratorytract infections (RTIs) are frequent and life-threatening diseases, accounting for several millions of deaths worldwide. RTIs implicate microorganisms, including viruses (influenza virus, coronavirus, respiratory syncytial virus (RSV)), bacteria (Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus and Bacillus anthracis) and fungi (Pneumocystis spp., Aspergillus spp. and very occasionally Candida spp.). The emergence of new pathogens, like the coronavirus SARS-CoV-2, and the substantial increase in drug resistance have highlighted the critical necessity to develop novel anti-infective molecules. In this context, antibodies (Abs) are becoming increasingly important in respiratory medicine and may fulfill the unmet medical needs of RTIs. However, development of Abs for treating infectious diseases is less advanced than for cancer and inflammatory diseases. Currently, only three Abs have been marketed for RTIs, namely, against pulmonary anthrax and RSV infection, while several clinical and preclinical studies are in progress. This article gives an overview of the advances in the use of Abs for the treatment of RTIs, based on the analysis of clinical studies in this field. It describes the Ab structure, function and pharmacokinetics, and discusses the opportunities offered by the various Ab formats, Ab engineering and co-treatment strategies. Including the most recent literature, it finally highlights the strengths, weaknesses and likely future trends of a novel anti-RTI Ab armamentarium.

Rat pancreatectomy combined with isoprenaline or uninephrectomy as models of diabetic cardiomyopathy or nephropathy.

Cardiovascular and renal complications are the predominant causes of morbidity and mortality amongst patients with diabetes. Development of novel treatments have been hampered by the lack of available animal models recapitulating the human disease. We hypothesized that experimental diabetes in rats combined with a cardiac or renal stressor, would mimic diabetic cardiomyopathy and nephropathy, respectively. Diabetes was surgically induced in male Sprague Dawley rats by 90% pancreatectomy (Px). Isoprenaline (Iso, 1 mg/kg, sc., 10 days) was administered 5 weeks after Px with the aim of inducing cardiomyopathy, and cardiac function and remodeling was assessed by echocardiography 10 weeks after surgery. Left ventricular (LV) fibrosis was quantified by Picro Sirius Red and gene expression analysis. Nephropathy was induced by Px combined with uninephrectomy (Px-UNx). Kidney function was assessed by measurement of glomerular filtration rate (GFR) and urine albumin excretion, and kidney injury was evaluated by histopathology and gene expression analysis. Px resulted in stable hyperglycemia, hypoinsulinemia, decreased C-peptide, and increased glycated hemoglobin (HbA1c) compared with sham-operated controls. Moreover, Px increased heart and LV weights and dimensions and caused a shift from α-myosin heavy chain (MHC) to ß-MHC gene expression. Isoprenaline treatment, but not Px, decreased ejection fraction and induced LV fibrosis. There was no apparent interaction between Px and Iso treatment. The superimposition of Px and UNx increased GFR, indicating hyperfiltration. Compared with sham-operated controls, Px-UNx induced albuminuria and increased urine markers of kidney injury, including neutrophil gelatinase-associated lipocalin (NGAL) and podocalyxin, concomitant with upregulated renal gene expression of NGAL and kidney injury molecule 1 (KIM-1). Whereas Px and isoprenaline separately produced clinical endpoints related to diabetic cardiomyopathy, the combination of the two did not accentuate disease development. Conversely, Px in combination with UNx resulted in several clinical hallmarks of diabetic nephropathy indicative of early disease development.

MAIT Cells Display a Specific Response to Type 1 IFN Underlying the Adjuvant Effect of TLR7/8 Ligands.

Mucosal-associated invariant T (MAIT) cells constitute a highly conserved subset of effector T cells with innate-like recognition of a wide array of bacteria and fungi in humans. Harnessing the potential of these cells could represent a major advance as a new immunotherapy approach to fight difficult-to-treat bacterial infections. However, despite recent advances in the design of potent agonistic ligands for MAIT cells, it has become increasingly evident that adjuvants are required to elicit potent antimicrobial effector functions by these cells, such as IFNγ production and cytotoxicity. Indeed, TCR triggering alone elicits mostly barrier repair functions in MAIT cells, whereas an inflammatory milieu is required to drive the antibacterial functions. Cytokines such as IL-7, IL-12 and IL-18, IL-15 or more recently type 1 IFN all display an apparently similar ability to synergize with TCR stimulation to induce IFNγ production and/or cytotoxic functions in vitro, but their mechanisms of action are not well established. Herein, we show that MAIT cells feature a build-in mechanism to respond to IFNα. We confirm that IFNα acts directly and specifically on MAIT cells and synergizes with TCR/CD3 triggering to induce maximum cytokine production and cytotoxic functions. We provide evidences suggesting that the preferential activation of the Stat4 pathway is involved in the high sensitivity of MAIT cells to IFNα stimulation. Finally, gene expression data confirm the specific responsiveness of MAIT cells to IFNα and pinpoints specific pathways that could be the target of this cytokine. Altogether, these data highlight the potential of IFNα-inducing adjuvants to maximize MAIT cells responsiveness to purified ligands in order to induce potent anti-infectious responses.

Automated Image Analyses of Glomerular Hypertrophy in a Mouse Model of Diabetic Nephropathy.

Background: Glomerular hypertrophy is a hallmark of kidney injury in metabolically induced renal diseases such as obesity-associated glomerulopathies and diabetic nephropathy (DN). Methods: Using light sheet fluorescent microscopy (LSFM) and 3D image analysis, we tested algorithms for automated and unbiased quantification of total glomerular numbers and individual glomerular volume in the uninephrectomized (UNx) db/db mouse model of DN. Results: At 6 weeks after surgery, db/db and UNx db/db mice showed increased urine albumin-to-creatinine ratio (ACR) compared with db/+ control mice. Before euthanasia, glomeruli were labeled in vivo by injecting tomato lectin. Whole-kidney LSFM 3D image analysis revealed that mean glomerular volume was significantly increased in UNx db/db mice compared with db/+ mice. Moreover, analysis of individual glomerular volume showed a shift in volume distribution toward larger glomeruli and thereby demonstrated additive effects of diabetes and UNx on induction of glomerular hypertrophy. The automatized quantification showed no significant differences in glomerular numbers among db/+, db/db, and UNx db/db mice. These data correlated with glomerular numbers as quantified by subsequent stereologic quantification. Conclusions: Overall, LSFM coupled with automated 3D histomorphometric analysis was demonstrated to be advantageous for unbiased assessment of glomerular volume and numbers in mouse whole-kidney samples. Furthermore, we showed that injection of fluorescently labeled lectin and albumin can be used as markers of nephron segments in the mouse kidneys, thus enabling functional assessment of kidney physiology, pathology, and pharmacology in preclinical rodent models of kidney disease.

Impact of sex on diabetic nephropathy and the renal transcriptome in UNx db/db C57BLKS mice.

Diabetic nephropathy (DN) is associated with albuminuria and loss of kidney function and is the leading cause of end-stage renal disease. Despite evidence of sex-associated differences in the progression of DN in human patients, male mice are predominantly being used in preclinical DN research and drug development. Here, we compared renal changes in male and female uninephrectomized (UNx) db/db C57BLKS mice using immunohistochemistry and RNA sequencing. Male and female UNx db/db mice showed similar progression of type 2 diabetes, as assessed by obesity, hyperglycemia, and HbA1c. Progression of DN was also similar between sexes as assessed by kidney and glomerular hypertrophy as well as urine albumin-to-creatinine ratio being increased in UNx db/db compared with control mice. In contrast, kidney collagen III and glomerular collagen IV were increased only in female UNx db/db as compared with respective control mice but showed a similar tendency in male UNx db/db mice. Comparison of renal cortex transcriptomes by RNA sequencing revealed 66 genes differentially expressed (p < .01) in male versus female UNx db/db mice, of which 9 genes were located on the sex chromosomes. In conclusion, male and female UNx db/db mice developed similar hallmarks of DN pathology, suggesting no or weak sex differences in the functional and structural changes during DN progression.