We aimed to understand the potential therapeutic and anti-inflammatory effects of the phosphodiesterase-4 (PDE4) inhibitor roflumilast in models of pulmonary infection caused by betacoronaviruses. Mice were infected intranasally with murine hepatitis virus (MHV-3) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Roflumilast was given to MHV-3-infected mice therapeutically at doses of 1 mg/kg or 10 mg/kg, or prophylactically at 10 mg/kg. In SARS-CoV-2-infected mice, roflumilast was given therapeutically at a dose of 10 mg/kg. Lung histopathology, chemokines (CXCL-1 and CCL2), cytokines (IL-1β, IL-6, TNF, IFN-γ, IL-10 and TGFβ), neutrophil immunohistochemical staining (Ly6G+ cells), macrophage immunofluorescence staining (F4/80+ cells), viral titration plaque assay, real-time PCR virus detection, and blood cell counts were examined. Therapeutic treatment with roflumilast at 10 mg/kg reduced lung injury in SARS-CoV-2 or MHV-3-infected mice without compromising viral clearance. In MHV-3-infected mice, reduced lung injury was associated with decreased chemokines levels, prevention of neutrophil aggregates and reduced macrophage accumulation in the lung tissue. However, the prophylactic treatment strategy with roflumilast increased lung injury in MHV-3-infected mice. Our findings indicate that therapeutic treatment with roflumilast reduced lung injury in MHV-3 and SARS-CoV-2 lung infections. Given the protection induced by roflumilast in inflammation, PDE4 targeting could be a promising therapeutic avenue worth exploring following severe viral infections of the lung.

Introduction:Immunotherapy has revolutionized cancer treatment by harnessing the immune system to enhance antitumor responses while minimizing off-target effects. Among the promising cancer-specific therapies, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted significant attention.Methods:Here, we developed an ionizable lipid nanoparticle (LNP) platform to deliver TRAIL mRNA (LNP-TRAIL) directly to the tumor microenvironment (TME) to induce tumor cell death. Our LNP-TRAIL was formulated via microfluidic mixing and the induction of tumor cell death was assessed in vitro. Next, we investigated the ability of LNP-TRAIL to inhibit colon cancer progression in vivo in combination with a TME normalization approach using Losartan (Los) or angiotensin 1-7 (Ang(1-7)) to reduce vascular compression and deposition of extracellular matrix in mice.Results:Our results demonstrated that LNP-TRAIL induced tumor cell death in vitro and effectively inhibited colon cancer progression in vivo, particularly when combined with TME normalization induced by treatment Los or Ang(1-7). In addition, potent tumor cell death as well as enhanced apoptosis and necrosis was found in the tumor tissue of a group treated with LNP-TRAIL combined with TME normalization.Discussion:Together, our data demonstrate the potential of the LNP to deliver TRAIL mRNA to the TME and to induce tumor cell death, especially when combined with TME normalization. Therefore, these findings provide important insights for the development of novel therapeutic strategies for the immunotherapy of solid tumors.

A safe and effective vaccine with long-term protection against SARS-CoV-2 variants of concern (VOCs) is a global health priority. Here, we develop lipid nanoparticles (LNPs) to provide safe and effective delivery of plasmid DNA (pDNA) and show protection against VOCs in female small animal models. Using a library of LNPs encapsulating unique barcoded DNA (b-DNA), we screen for b-DNA delivery after intramuscular administration. The top-performing LNPs are further tested for their capacity of pDNA uptake in antigen-presenting cells in vitro. The lead LNP is used to encapsulate pDNA encoding the HexaPro version of SARS-CoV-2 spike (LNP-HPS) and immunogenicity and protection is tested in vivo. LNP-HPS elicit a robust protective effect against SARS-CoV-2 Gamma (P.1), correlating with reduced lethality, decreased viral load in the lungs and reduced lung damage. LNP-HPS induce potent humoral and T cell responses against P.1, and generate high levels of neutralizing antibodies against P.1 and Omicron (B.1.1.529). Our findings indicate that the protective efficacy and immunogenicity elicited by LNP-HPS are comparable to those achieved by the approved COVID-19 vaccine from Biontech/Pfizer in animal models. Together, these findings suggest that LNP-HPS hold great promise as a vaccine candidate against VOCs.

The pathophysiology of post-traumatic brain injury (TBI) behavioral and cognitive changes is not fully understood, especially in its mild presentation. We designed a weight drop TBI model in mice to investigate the role of neuroinflammation in behavioral and cognitive sequelae following mild TBI. C57BL/6 mice displayed depressive-like behavior at 72 h after mild TBI compared with controls, as indicated by a decrease in the latency to first immobility and climbing time in the forced swim test. Additionally, anxiety-like behavior and hippocampal-associated spatial learning and memory impairment were found in the elevated plus maze and in the Barnes maze, respectively. Levels of a set of inflammatory mediators and neurotrophic factors were analyzed at 6 h, 24 h, 72 h, and 30 days after injury in ipsilateral and contralateral hemispheres of the prefrontal cortex and hippocampus. Principal components analysis revealed two principal components (PC), which represented 59.1% of data variability. PC1 (cytokines and chemokines) expression varied between both hemispheres, while PC2 (neurotrophic factors) expression varied only across the investigated brain areas. Our model reproduces mild TBI-associated clinical signs and pathological features and might be a valuable tool to broaden the knowledge regarding mild TBI pathophysiology as well as to test potential therapeutic targets.

Systemic arterial hypertension (SAH) is a major risk factor for several secondary diseases, especially cardiovascular and renal conditions. SAH has a high prevalence worldwide, and its precise and early recognition is important to prevent the development of secondary outcomes. In this field, the study of biomarkers represents an important approach to diagnosing and predicting the disease and its associated conditions. The use of biomarkers in hypertension and hypertension-related disorders, such as ischemic stroke, intracerebral hemorrhage, transient ischemic attack, acute myocardial infarction, angina pectoris and chronic kidney disease, are discussed in this review. Establishing a potential pool of biomarkers may contribute to a non-invasive and improved approach for their diagnosis, prognosis, risk assessment, therapy management and pharmacological responses to a therapeutic intervention to improve patients' quality of life and prevent unfavorable outcomes.

CXCL12 is a key chemokine implicated in neuroinflammation, particularly during Zika virus (ZIKV) infection. Specifically, CXCL12 is upregulated in circulating cells of ZIKV infected patients. Here, we developed a lipid nanoparticle (LNP) to deliver siRNA in vivo to assess the impact of CXCL12 silencing in the context of ZIKV infection. The biodistribution of the LNP was assessed in vivo after intravenous injection using fluorescently tagged siRNA. Next, we investigated the ability of the developed LNP to silence CXCL12 in vivo and assessed the resulting effects in a murine model of ZIKV infection. The LNP encapsulating siRNA significantly inhibited CXCL12 levels in the spleen and induced microglial activation in the brain during ZIKV infection. This activation was evidenced by the enhanced expression of iNOS, TNF-α, and CD206 within microglial cells. Moreover, T cell subsets exhibited reduced secretion of IFN-ɣ and IL-17 following LNP treatment. Despite no observable alteration in viral load, CXCL12 silencing led to a significant reduction in type-I interferon production compared to both ZIKV-infected and uninfected groups. Furthermore, we found grip strength deficits in the group treated with siRNA-LNP compared to the other groups. Our data suggest a correlation between the upregulated pro-inflammatory cytokines and the observed decrease in strength. Collectively, our results provide evidence that CXCL12 silencing exerts a regulatory influence on the immune response in the brain during ZIKV infection. In addition, the modulation of T-cell activation following CXCL12 silencing provides valuable insights into potential protective mechanisms against ZIKV, offering novel perspectives for combating this infection.

The use of implantable biomaterials to replace physiological and anatomical functions has been widely investigated in the clinic. However, the selection of biomaterials is crucial for long-term function, and the implantation of certain biomaterials can cause inflammatory and fibrotic processes, triggering a foreign body reaction that leads to loss of function and consequent need for removal. Specifically, the Wnt signaling pathway controls the healing process of the human body, and its dysregulation can result in inflammation and fibrosis, such as in peritoneal fibrosis. Here, we assessed the effects of daily oral administration of a Wnt pathway inhibitor complex (CD:LGK974) to reduce the inflammatory, fibrotic, and angiogenic processes caused by intraperitoneal implants. CD:LGK974 significantly reduced the infiltration of immune cells and release of inflammatory cytokines in the implant region compared to the control groups. Furthermore, CD:LGK974 inhibited collagen deposition and reduced the expression of pro-fibrotic α-SMA and TGF-β1, confirming fibrosis reduction. Finally, the CD:LGK974 complex decreased VEGF levels and both the number and area of blood vessels formed, suggesting decreased angiogenesis. This work introduces a potential new application of the Wnt inhibitor complex to reduce peritoneal fibrosis and the rejection of implants at the intraperitoneal site, possibly allowing for longer-term functionality of existing clinical biomaterials.

Cancer cells are embedded within the tissue and interact dynamically with its components during cancer progression. Understanding the contribution of cellular components within the tumor microenvironment is crucial for the success of therapeutic applications. Here, we reveal the presence of perivascular GFAP+/Plp1+ cells within the tumor microenvironment. Using in vivo inducible Cre/loxP mediated systems, we demonstrated that these cells derive from tissue-resident Schwann cells. Genetic ablation of endogenous Schwann cells slowed down tumor growth and angiogenesis. Schwann cell-specific depletion also induced a boost in the immune surveillance by increasing tumor-infiltrating anti-tumor lymphocytes, while reducing immune-suppressor cells. In humans, a retrospective in silico analysis of tumor biopsies revealed that increased expression of Schwann cell-related genes within melanoma was associated with improved survival. Collectively, our study suggests that Schwann cells regulate tumor progression, indicating that manipulation of Schwann cells may provide a valuable tool to improve cancer patients' outcomes.

Hematopoietic stem cells are the most illustrious inhabitants of the bone marrow. Direct visualization of endogenous hematopoietic stem cells in this niche is essential to study their functions. Until recently this was not possible in live animals. Recent studies, using state-of-the-art technologies, including sophisticated in vivo inducible genetic approaches in combination with two-photon laser scanning microscopy, allow the follow-up of endogenous hematopoietic stem cells' behavior in their habitat. Strikingly, the new findings reveal that quiescent hematopoietic stem cells are more mobile than previously thought, and link their retained steady state within the niche to a mobile behavior. The arising knowledge from this research will be critical for the therapy of several hematological diseases. Here, we review recent progress in our understanding of hematopoietic stem cell biology in their niches.

Diagnosis and prognosis of breast cancer is based on disease staging identified through histopathological and molecular biology techniques. Animal models are used to gain mechanistic insights into the development of breast cancer. C(3)1-TAg is a genetically engineered mouse model that develops mammary cancer. However, carcinogenesis caused by this transgene was characterized in the Friend Virus B (FVB) background. As most genetic studies are done in mice with C57BL/6 J background, we aimed to define the histological alterations in C3(1)-TAg C57BL/6 J animals. Our results showed that C3(1)-TAg animals with C57BL/6 J background develop solid-basaloid adenoid cystic carcinomas with increased fibrosis, decreased area of adipocytes, and a high proliferative index, which are triple-negative for progesterone, estrogen, and human epidermal growth factor receptor 2 (HER2) receptors. Our results also revealed that tumor development is slower in the C57BL/6 J background when compared with the FVB strain, providing a better model to study the different stages in breast cancer progression.

Multiple infectious diseases lead to impaired lung function. Revealing the cellular mechanisms involved in this impairment is crucial for the understanding of how the lungs shift from a physiologic to a pathologic state in each specific condition. In this context, we explored the pathogenesis of Paracoccidioidomycosis, which affects pulmonary functioning. The presence of cells expressing Nestin-GFP has been reported in different tissues, and their roles as tissue-specific progenitors have been stablished in particular organs. Here, we explored how Nestin-GFP+ cells are affected after lung infection by Paracoccidioides brasiliensis, a model of lung granulomatous inflammation with fibrotic outcome. We used Nestin-GFP transgenic mice, parabiosis surgery, confocal microscopy and flow cytometry to investigate the participation of Nestin-GFP+ cells in Paracoccidioides brasiliensis pathogenesis. We revealed that these cells increase in the lungs post-Paracoccidioides brasiliensis infection, accumulating around granulomas. This increase was due mainly to Nestin-GPF+ cells derived from the blood circulation, not associated to blood vessels, that co-express markers suggestive of hematopoietic cells (Sca-1, CD45 and CXCR4). Therefore, our findings suggest that circulating Nestin-GFP+ cells participate in the Paracoccidioides brasiliensis pathogenesis in the lungs.

The tumour mass is composed not only of heterogeneous neoplastic cells, but also a variety of other components that may affect cancer cells behaviour. The lack of detailed knowledge about all the constituents of the tumour microenvironment restricts the design of effective treatments. Nerves have been reported to contribute to the growth and maintenance of numerous tissues. The effects of sensory innervations on tumour growth remain unclear. Here, by using state-of-the-art techniques, including Cre/loxP technologies, confocal microscopy, in vivo-tracing and chemical denervation, we revealed the presence of sensory nerves infiltrating within the melanoma microenvironment, and affecting cancer progression. Strikingly, melanoma growth in vivo was accelerated following genetic ablation or chemical denervation of sensory nerves. In humans, a retrospective analysis of melanoma patients revealed that increased expression of genes related to sensory nerves in tumours was associated with better clinical outcomes. These findings suggest that sensory innervations counteract melanoma progression. The emerging knowledge from this research provides a novel target in the tumour microenvironment for therapeutic benefit in cancer patients.

Joint pain is a distressing symptom of arthritis, and it is frequently persistent even after treatments which reduce local inflammation. Continuous production of algogenic factors activate/sensitize nociceptors in the joint structures and contribute to persistent pain, a challenging and difficult condition to treat. TNF is a crucial cytokine for the pathogenesis of several rheumatic diseases, and its inhibition is a mainstay of treatment to control joint symptoms, including pain. Here, we sought to investigate the inflammatory changes and the role of TNF in dorsal root ganglia (DRG) during persistent hypernociception after the resolution of acute joint inflammation. Using a model of antigen-induced arthritis, the peak of joint inflammation occurred 12-24 h after local antigen injection and was characterized by an intense influx of neutrophils, pro-inflammatory cytokine production, and joint damage. We found that inflammatory parameters in the joint returned to basal levels between 6 and 8 days after antigen-challenge, characterizing the resolving phase of joint inflammation. Mechanical hyperalgesia was persistent up to 14 days after joint insult. The persistent nociception was associated with the inflammatory status of DRG after cessation of acute joint inflammation. The late state of neuroinflammation in the ipsilateral side was evidenced by gene expression of TNF, TNFR2, IL-6, IL-1β, CXCL2, COX2, and iNOS in lumbar DRG (L3-L5) and leukocyte adhesion in the lumbar intumescent vessels between days 6 and 8. Moreover, there were signs of resident macrophage activation in DRG, as evidenced by an increase in Iba1-positive cells. Intrathecal or systemic injection of etanercept, an agent clinically utilized for TNF neutralization, at day 7 post arthritis induction, alleviated the persistent joint hyperalgesia by specific action in DRG. Our data suggest that neuroinflammation in DRG after the resolution of acute joint inflammation drives continuous neural sensitization resulting in persistent joint nociception in a TNF-dependent mechanism.

Niches are specialized tissue microenvironments that control stem cells functioning. The bone marrow mesenchymal stem cell niche defines a location within the marrow in which mesenchymal stem cells are retained and produce new cells throughout life. Deciphering the signaling mechanisms by which the niche regulates stem cell fate will facilitate the use of these cells for therapy. Recent studies, by using state-of-the-art methodologies, including sophisticated in vivo inducible genetic techniques, such as lineage-tracing Cre/loxP mediated systems, in combination with pharmacological inhibition, provide evidence that sensory neuron is an important component of the bone marrow mesenchymal stem cell niche. Strikingly, knockout of a specific receptor in sensory neurons blocked stem cell function in the bone marrow. The knowledge arising from these discoveries will be crucial for stem cell manipulation in the future. Here, we review recent progress in our understanding of sensory nerves biology in the stem cell niche.

Spinal cord injury results in locomotor impairment attributable to the formation of an inhibitory fibrous scar, which prevents axonal regeneration after trauma. The scarcity of knowledge about the molecular and cellular mechanisms involved in scar formation after spinal cord lesion impede the design of effective therapies. Recent studies, by using state-of-the-art technologies, including genetic tracking and blockage of pericytes in combination with optogenetics, reveal that pericyte blockage facilitates axonal regeneration and neuronal integration into the local neural circuitry. Strikingly, a pericyte subset is essential during scarring after spinal cord injury, and its arrest results in motor performance improvement. The arising knowledge from current research will contribute to novel approaches to develop therapies for spinal cord injury. We review novel advances in our understanding of pericyte biology in the spinal cord.

Spinal cord injury results in locomotor impairment attributable to the formation of an inhibitory fibrous scar, which prevents axonal regeneration after trauma. The scarcity of knowledge about the molecular and cellular mechanisms involved in scar formation after spinal cord lesion impede the design of effective therapies. Recent studies, by using state-of-the-art technologies, including genetic tracking and blockage of pericytes in combination with optogenetics, reveal that pericyte blockage facilitates axonal regeneration and neuronal integration into the local neural circuitry. Strikingly, a pericyte subset is essential during scarring after spinal cord injury, and its arrest results in motor performance improvement. The arising knowledge from current research will contribute to novel approaches to develop therapies for spinal cord injury. We review novel advances in our understanding of pericyte biology in the spinal cord.

In mammals, new neurons can be generated from neural stem cells in specific regions of the adult brain. Neural stem cells are characterized by their abilities to differentiate into all neural lineages and to self-renew. The specific microenvironments regulating neural stem cells, commonly referred to as neurogenic niches, comprise multiple cell populations whose precise contributions are under active current exploration. Understanding the cross-talk between neural stem cells and their niche components is essential for the development of therapies against neurological disorders in which neural stem cells function is altered. In this review, we describe and discuss recent studies that identified novel components in the neural stem cell niche. These discoveries bring new concepts to the field. Here, we evaluate these recent advances that change our understanding of the neural stem cell niche heterogeneity and its influence on neural stem cell function.

Wound healing is a complex dynamic physiological process in response to cutaneous destructive stimuli that aims to restore the cutaneous' barrier role. Deciphering the underlying mechanistic details that contribute to wound healing will create novel therapeutic strategies for skin repair. Recently, by using state-of-the-art technologies, it was revealed that the cutaneous microbiota interact with skin immune cells. Strikingly, commensal Staphylococcus epidermidis-induced CD8+ T cells induce re-epithelization of the skin after injury, accelerating wound closure. From a drug development perspective, the microbiota may provide new therapeutic candidate molecules to accelerate skin healing. Here, we summarize and evaluate recent advances in the understanding of the microbiota in the skin microenvironment.