Early Inflammatory Phase Research Articles

Evaluation of heterotopic ossification maturity using SPECT/CT and PET/CT in preclinical model

BackgroundHeterotopic ossification (HO) is the formation of bone within soft tissue where bone normally does not exist. In general, it is characterized by highly active tissue with high bone turnover and rapid bone formation. It is of an utmost importance to precisely identify and accurately diagnose the maturity of HO as early surgical intervention may result in its recurrence. The objective of this work is the experimental evaluation of HO maturity stage using advanced noninvasive nuclear medicine techniques. The use of PET radiopharmaceuticals may result in a more specific diagnosis between the phases due to their higher sensitivity and better resolution compared to bone scan. Method8-week-old Balb/c male mice underwent dual injury procedure, tenotomy and concurrent burn injury on the left side, to induce HO. The progression of HO was monitored by SPECT/CT and PET/CT weekly imaging with 99mTc-MDP, [18F]NaF and [18F]FDG for up to 16 weeks. ResultsThere was a statistically significant increase of [18F]FDG uptake from week 1 to 2 and from week 2 to 6 with p values of 0.01 and 0.005; respectively, while there was a statistically significant decrease from week 7 to 14 with a p value of 0.008. There was a statistically significant increase of [18F]NaF uptake from week 2 to 5 and statistically significant decrease between weeks 7 and 14 with p values of 0.016 and 0.003; respectively. As for 99mTc-MDP, the increase in the uptake from week 1 to 2 and from week 2 to 5 were not statistically significant with p values of 0.15 and 0.19; respectively. The decrease of uptake between week 7 and 14 was not statistically significant with a p value of 0.08. ConclusionBased on these findings, the use of noninvasive nuclear imaging modalities may assist in distinguishing between the immature and mature phases. The uptake of mainly [18F]FDG may indicate the early inflammatory phase, while the uptake of both [18F]FGD and [18F]NaF may suggest the immature phase, and an uptake of mainly [18F]NaF may indicate the maturity phase of HO.

Metabolomics Reveals Disturbed Amino Acid Metabolism During Different Stages of RA in Collagen-Induced Arthritis Mice.

Rheumatoid arthritis (RA) is an autoimmune disease featured by chronic synovitis and progressive joint damage. Early treatment before the onset of clinical symptoms (also known as the pre-RA stage) may slow or stop the progression of the disease. We sought to discover the dynamic metabolic changes during the evolution of collagen-induced arthritis (CIA) to better characterize the disease stages. Untargeted metabolomics analysis using gas chromatography-mass spectrometry revealed that the metabolic profiles of CIA mice gradually differed from that of the control group with the progression of the disease. During the induction phase, the CIA group showed some metabolic alterations in galactose metabolism, arginine biosynthesis, tricarboxylic acid cycle (TCA cycle), pyruvate metabolism, and starch/sucrose metabolism. During the early inflammatory phase, no joint swelling was observed in CIA mice, and metabolites changed mainly involving amino acid metabolism (arginine biosynthesis, arginine/proline metabolism, phenylalanine/tyrosine/tryptophan biosynthesis), and glutathione metabolism. During the peak inflammatory phase, severe arthritis symptoms were observed in CIA mice, and there were more extensive metabolic alterations in valine/leucine/isoleucine biosynthesis, phenylalanine/tyrosine/tryptophan biosynthesis, TCA cycle, galactose metabolism, and arginine biosynthesis. Moreover, the reduction of specific amino acids, such as glycine, serine, and proline, during the early stages may result in an imbalance in macrophage polarization and enhance the inflammatory response in CIA mice. Our study confirmed that specific perturbations in amino acid metabolism have occurred in CIA mice prior to the onset of joint symptoms, which may be related to autoimmune disorders. The findings could provide insights into the metabolic mechanism and the diagnosis of pre-RA.

The Impact of PAD4-dependent Neutrophil Extracellular Trap Formation on the Early Development of Intestinal Fibrosis in Crohn's Disease.

During early phases of inflammation, activated neutrophils extrude neutrophil extracellular traps (NETs) in a PAD4-dependent manner, aggravating tissue injury and remodelling. In this study, we investigated the potential pro-fibrotic properties and signalling of NETs in Crohn's disease (CD). NETs and activated fibroblasts were labelled on resected ileum from CD patients by multiplex immunofluorescence staining. NETs-treated human primary intestinal fibroblasts were analysed by bulk RNA-sequencing to uncover cell signalling pathways, and by high-throughput imaging to assess collagen production and migratory activity. Consequentially, TLR2/NF-kB pathway was evaluated by transfection of CCD-18Co fibroblasts with NF-kB-luciferase reporter plasmid, incorporating C29 to block TLR2 signalling. A chronic DSS mouse model was used to define the specific role of PAD4 deletion in neutrophils (MRP8-Cre, Pad4fl/fl). Immunofluorescence showed spatial co-localisation of NETs and activated fibroblasts in ileal ulcerations of CD patients. Transcriptomic analysis revealed upregulation of pro-fibrotic genes and activation of TLR-signalling pathways in NETs-treated fibroblasts. NETs treatment induced fibroblast proliferation, diminished migratory capability, and increased collagen release. Transfection experiments indicated a substantial increase in NF-kB expression with NETs, whereas C29 led to decreased expression and release of collagen. In line, a significantly reduction in collagen content was observed in the colon of MRP8-Cre, Pad4fl/fl mice subjected to chronic DSS colitis. NETs potentially serve as an initial stimulus for pathological activation of fibroblasts within the intestine via the TLR2/NF-kB pathway. Given their early involvement in inflammation, inhibition of PAD4 might offer a strategy to modulate both inflammation and fibrogenesis in CD.

Sequential management of burn wound healing stages through biointelligence-inspired platelet extracellular vesicle-encapsulated photodynamic diferuloylmethane.

The process of wound healing is a complex, multi-phase phenomenon crucial for optimal tissue regeneration. Traditional drug delivery systems often target specific phases of wound repair, neglecting the dynamic interplay among the stages. This limitation highlights the need for comprehensive delivery systems that cater to the holistic needs of wound healing, enhancing tissue regeneration efficiency. Herein, we explored the utility of platelet-derived extracellular vesicles (pEVs) as carriers for the phototherapeutic diferuloylmethane (DIF), resulting in a formulation termed DIF@pEVs, which is designed to sequentially address the distinct phases of wound healing. Initially, upon exposure to light, administered DIF@pEVs generate photodynamic therapy-derived reactive oxygen species during the early inflammatory phase. This generation of ROS aims to modulate the inflammatory response, induce the protective mechanisms of heat shock proteins, and kickstart the tissue regeneration process. Following this initial phase, the remaining DIF and pEVs persist in promoting tissue repair and regeneration. Ultimately, it reduces inflammation, speeds up the healing process, and promotes vascular and follicular formation in a model of burn wound skin damage, thereby supporting skin regeneration. The deployment of DIF@pEVs represents an advancement in regenerative medicine, providing a precise, versatile approach to fostering regeneration across a wide range of clinical scenarios.

The Diagnostic and Prognostic Values of Soluble CD14 Subtype (Presepsin) in Early Detection of Early Onset Neonatal Sepsis in Preterm Newborns

Abstract Background The diagnosis of “suspected sepsis” in neonates in the NICU is one of the most common and challenging diagnoses due to the nonspecific signs of sepsis. therefore, laboratory investigation is necessary in cases of doubt or if there are risk factors. A promising biomarker: Presepsin (P-SEP), or Soluble Cluster of Differentiation 14 Subtype (sCD14-ST) is generated as a part of the body’s response to bacterial infection. It is effective in the early phase of inflammation, and it is detected as an early marker of sepsis in neonates. Aim and Objectives to evaluate the diagnostic value of presepsin in early neonatal sepsis and prognostic value for severity, mortality and hospital stay length. Subjects and Methods This prospective observational study include two groups, one group included 36 neonates with risk factors for early-onset sepsis, along with 20 uninfected neonates as the control group. Both groups were subjected to the calculation of Rodwell score, SNAP II score, serum presepsin levels, CRP measurement, and blood culture and assessed for clinical severity, mortality, and hospital stay length. Result Serum baseline presepsin was significantly elevated in sepsis group (P < 0.001), Presepsin showed the best diagnostic performance over Rodwell score (AUC 0.989; sensitivity of 93.8% and specificity of 95%). Presepsin levels on day 3 were significantly elevated in non-survived patients (P < 0.001), with better predictive performance for 28-day mortality and rapid progression to death than SNAP II score (AUC 0.880; sensitivity of 83.3% and specificity of 87.5%). Conclusion presepsin day 1 was useful diagnostic tools in culture proven early onset neonatal sepsis, presepsin at day 3 could be a good predictor for gram negative infections, 28-day mortality, and sepsis severity.

Repeated social defeat stress differently affects arthritis-associated hypersensitivity in male and female mice

Chronic stress enhances the risk of neuropsychiatric disorders and contributes to the aggravation and chronicity of pain. The development of stress-associated diseases, including pain, is affected by individual vulnerability or resilience to stress, although the mechanisms remain elusive. We used the repeated social defeat stress model promoting susceptible and resilient phenotypes in male and female mice and induced knee mono-arthritis to investigate the impact of stress vulnerability on pain and immune system regulation. We analyzed different pain-related behaviors, measured blood cytokine and immune cell levels, and performed histological analyses at the knee joints and pain/stress-related brain areas. Stress susceptible male and female mice showed prolonged arthritis-associated hypersensitivity. Interestingly, hypersensitivity was exacerbated in male but not female mice. In males, stress promoted transiently increased neutrophils and Ly6Chigh monocytes, lasting longer in susceptible than resilient mice. While resilient male mice displayed persistently increased levels of the anti-inflammatory interleukin (IL)-10, susceptible mice showed increased levels of the pro-inflammatory IL-6 at the early- and IL-12 at the late arthritis stage. Although joint inflammation levels were comparable among groups, macrophage and neutrophil infiltration was higher in the synovium of susceptible mice. Notably, only susceptible male mice, but not females, presented microgliosis and monocyte infiltration in the prefrontal cortex at the late arthritis stage. Blood Ly6Chigh monocyte depletion during the early inflammatory phase abrogated late-stage hypersensitivity and the associated histological alterations in susceptible male mice.Thus, recruitment of blood Ly6Chigh monocytes during the early arthritis phase might be a key factor mediating the persistence of arthritis pain in susceptible male mice. Alternative neuro-immune pathways that remain to be explored might be involved in females.

Single cell transcriptomic analyses reveal diverse and dynamic changes of distinct populations of lung interstitial macrophages in hypoxia-induced pulmonary hypertension.

Hypoxia is a common pathological driver contributing to various forms of pulmonary vascular diseases leading to pulmonary hypertension (PH). Pulmonary interstitial macrophages (IMs) play pivotal roles in immune and vascular dysfunction, leading to inflammation, abnormal remodeling, and fibrosis in PH. However, IMs' response to hypoxia and their role in PH progression remain largely unknown. We utilized a murine model of hypoxia-induced PH to investigate the repertoire and functional profiles of IMs in response to acute and prolonged hypoxia, aiming to elucidate their contributions to PH development. We conducted single-cell transcriptomic analyses to characterize the repertoire and functional profiles of murine pulmonary IMs following exposure to hypobaric hypoxia for varying durations (0, 1, 3, 7, and 21 days). Hallmark pathways from the mouse Molecular Signatures Database were utilized to characterize the molecular function of the IM subpopulation in response to hypoxia. Our analysis revealed an early acute inflammatory phase during acute hypoxia exposure (Days 1-3), which was resolved by Day 7, followed by a pro-remodeling phase during prolonged hypoxia (Days 7-21). These phases were marked by distinct subpopulations of IMs: MHCIIhiCCR2+EAR2+ cells characterized the acute inflammatory phase, while TLF+VCAM1hi cells dominated the pro-remodeling phase. The acute inflammatory phase exhibited enrichment in interferon-gamma, IL-2, and IL-6 pathways, while the pro-remodeling phase showed dysregulated chemokine production, hemoglobin clearance, and tissue repair profiles, along with activation of distinct complement pathways. Our findings demonstrate the existence of distinct populations of pulmonary interstitial macrophages corresponding to acute and prolonged hypoxia exposure, pivotal in regulating the inflammatory and remodeling phases of PH pathogenesis. This understanding offers potential avenues for targeted interventions, tailored to specific populations and distinct phases of the disease. Moreover, further identification of triggers for pro-remodeling IMs holds promise in unveiling novel therapeutic strategies for pulmonary hypertension.

ScRNA-Seq: First Atlas and Cellular Landscape of Lacrimal Sac: Implications in Primary Acquired Nasolacrimal Duct Obstruction Pathogenesis.

The aim of this study was to describe the transcriptional changes of individual cellular components in the lacrimal sac in patients with primary acquired nasolacrimal duct obstruction (PANDO) and attempt to construct the first lacrimal sac cellular atlas to elucidate the potential mechanisms that may drive the disease pathogenesis. Lacrimal sac samples were obtained intra-operatively during the endoscopic dacryocystorhinostomy (EnDCR) procedure from five patients. Single-cell RNA sequencing was performed to analyze each individual cell population including epithelial and immune cells during the early inflammatory and late inflammatory phases of the disease. Eleven cell types were identified among 25,791 cells. T cells and B cells were the cell populations with the greatest variation in cell numbers between the two phases and were involved in immune response and epithelium migration-related pathways. The present study showed that epithelial cells highly expressed the genes of senescence-associated secretory phenotype (SASP) and were involved in influencing the inflammation, neutrophil chemotaxis, and migration during the late inflammatory stage. Enhanced activity of CXCLs-CXCRs between the epithelial cells and neutrophils was noted by the cell-cell communication analysis and is suspected to play a role in inflammation by recruiting more neutrophils. The study presents a comprehensive single-cell landscape of the lacrimal sac cells in different phases of PANDO. The contribution of T cells, B cells, and epithelial cells to the inflammatory response, and construction of the intercellular signaling networks between the cells within the lacrimal sac has further enhanced the present understanding of the PANDO pathogenesis.

NLRP3 inflammasome mediates abnormal epithelial regeneration and distal lung remodeling in silica‑induced lung fibrosis.

NOD-like receptor protein 3 (NLRP3) inflammasome is closely related to silica particle‑induced chronic lung inflammation but its role in epithelial remodeling, repair and regeneration in the distal lung during development of silicosis remains to be elucidated. The present study aimed to determine the effects of the NLRP3 inflammasome on epithelial remodeling and cellular regeneration and potential mechanisms in the distal lung of silica‑treated mice at three time points. Pulmonary function assessment, inflammatory cell counting, enzyme‑linked immunosorbent assay, histological and immunological analyses, hydroxyproline assay and western blotting were used in the study. Single intratracheal instillation of a silica suspension caused sustained NLRP3 inflammasome activation in the distal lung. Moreover, a time‑dependent increase in airway resistance and a decrease in lung compliance accompanied progression of pulmonary fibrosis. In the terminal bronchiole, lung remodeling including pyroptosis (membrane‑distributed GSDMD+), excessive proliferation (Ki67+), mucus overproduction (mucin 5 subtype AC and B) and epithelial‑mesenchymal transition (decreased E‑Cadherin+ and increased Vimentin+), was observed by immunofluorescence analysis. Notably, aberrant spatiotemporal expression of the embryonic lung stem/progenitor cell markers SOX2 and SOX9 and ectopic distribution of bronchioalveolar stem cells were observed in the distal lung only on the 7th day after silica instillation (the early inflammatory phase of silicosis). Western blotting revealed that the Sonic hedgehog/Glioma‑associated oncogene (Shh/Gli) and Wnt/β‑catenin pathways were involved in NLRP3 inflammasome activation‑mediated epithelial remodeling and dysregulated regeneration during the inflammatory and fibrotic phases. Overall, sustained NLRP3 inflammasome activation led to epithelial remodeling in the distal lung of mice. Moreover, understanding the spatiotemporal profile of dysregulated epithelial repair and regeneration may provide a novel therapeutic strategy for inhalable particle‑related chronic inflammatory and fibrotic lung disease.

Wound Healing Improvement by Polymer Surgical Suture Coated with Tannic Acid

A surgical suture is a vital medical device to uphold wounded tissue during surgery. However, the application to improve tissue healing must still be developed. This study offered to solve this problem by incorporating tannic acid (TA) into polydioxanone (PDS) sutures. The PDS suture was immersed in TA complexing with iron (III) solutions in pH 8.0 solution. The engineered suture was successful, as revealed by the OH peak of TA in FTIR spectra. Furthermore, it was confirmed by TA particle appearance under Scanning Electron Microscopy. Contact angle measurement showed better wettability in biological mimicking solutions, such as water, serum, and saline solution. Thereby, it improved in vitro wound healing activity. Moreover, our bioinformatics data showed that TA enhanced the wound healing process by addressing multiple targets in the early phase of inflammation. For instance, it induced platelets to reduce bleeding at the early wound healing stage, activated T cells at the inflammation stage, accelerated epidermis cell proliferation, and activated fibroblast as a key player in wound healing. Due to multiple wound healing targets, our suture system was expected to be emphasized in clinical applications. HIGHLIGHTS The engineered surgical suture was fabricated by modifying the surface of surgical suture with tannic acid (TA) through iron (III) complex formation in basic condition The modified PDS suture had a high wettability degree of water, saline, and serum, representing the biological fluid, which was expected to have better biocompatibility Based on bioinformatics analysis, TA revealed multiple targets to stop bleeding by activating platelet through FYN, CD4, and CD8 T cells, induced epithelial cells proliferation through Epidermal Growth Factor Receptor (EGFR), and activated many transcription and migration factors of fibroblast GRAPHICAL ABSTRACT

Mmp12 Is Translationally Regulated in Macrophages during the Course of Inflammation

Despite the importance of rapid adaptive responses in the course of inflammation and the notion that post-transcriptional regulation plays an important role herein, relevant translational alterations, especially during the resolution phase, remain largely elusive. In the present study, we analyzed translational changes in inflammatory bone marrow-derived macrophages upon resolution-promoting efferocytosis. Total RNA-sequencing confirmed that apoptotic cell phagocytosis induced a pro-resolution signature in LPS/IFNγ-stimulated macrophages (Mϕ). While inflammation-dependent transcriptional changes were relatively small between efferocytic and non-efferocytic Mϕ; considerable differences were observed at the level of de novo synthesized proteins. Interestingly, translationally regulated targets in response to inflammatory stimuli were mostly downregulated, with only minimal impact of efferocytosis. Amongst these targets, pro-resolving matrix metallopeptidase 12 (Mmp12) was identified as a translationally repressed candidate during early inflammation that recovered during the resolution phase. Functionally, reduced MMP12 production enhanced matrix-dependent migration of Mϕ. Conclusively, translational control of MMP12 emerged as an efficient strategy to alter the migratory properties of Mϕ throughout the inflammatory response, enabling Mϕ migration within the early inflammatory phase while restricting migration during the resolution phase.

Monocyte priming during acute myocardial infarction

Abstract Introduction Purpose Myocardial infarction (MI) inflicts a sterile wound on the heart which recruits hundreds of thousands of monocytes from the blood. The bone marrow (BM) responds to cardiac ischemia with an increase in monocyte production, hence meeting the high demand of these cells. Apart from this quantitative response, little is known whether the BM supplies qualitatively different monocytes during the course of MI. Methods & Results We isolated BM Ly6Chigh monocytes from non-infarcted mice and from mice which were infarcted 72h prior (surgically induced by permanent coronary ligation) and contrasted monocytic transcriptomes using RNA sequencing. We found 672 differentially expressed genes (DEG) of which 289 were up- and 383 down-regulated (log2FoldChange³±1.0, adjusted p<0.05). Since most DEG were enriched in "cell adhesion and recruitment" pathways, we tested the functional relevance of our findings in the context of monocyte recruitment to the heart using competitive adoptive transfer experiments. Our data revealed that the BM provides monocytes that are primed for better recruitment (relative recruitment: 56.1% 12h after MI vs. 43.9% steady state, p<0.01) at the early inflammatory phase following coronary ligation (12h after MI). In contrast, monocytes displayed worse recruitment capacities (relative recruitment: 30.4% 72h after MI vs. 65.8% steady state, p<0.01) at the end of the inflammatory phase (72h after MI). We next sought to identify key players in this BM-controlled cardiac recruitment process. Our RNA sequencing data revealed that – in monocytes with poor recruitment capacities – the cell adhesion molecule CD209 was among the most significantly downregulated genes at 72h after MI over steady state. To investigate the molecular role of CD209 in the process of leukocyte recruitment to the myocardium during MI, we cloned human CD209 from a cDNA library into an overexpression plasmid system. After ectopic overexpression of CD209, THP-1 cells (a cell line derived from immortalized human blood monocytes) showed enhanced adhesion to endothelial cells (HUVECs) compared to THP-1 cells under control conditions. Finally, we infarcted wild type (CD209+/+) and CD209 knockout (CD209-/-) mice and found decreased numbers of Ly6Chigh monocytes in infarcted hearts of CD209-/- mice in comparison to CD209+/+ mice (491+52 vs. 915+180 Ly6Chigh monocytes/mg infarct tissue, mean+SEM, p<0.05) while numbers of blood Ly6Chigh monocytes were unchanged. Conclusions The BM responds to MI and provides an altered monocyte repertoire in terms of numbers and function and hence remotely controls monocyte/macrophage accumulation in the ischemic heart. Consequently, the heart–bone marrow axis may represent a novel therapeutic target to beneficially modify post-MI cardiac remodeling, a major contributor of MI-related morbidity and mortality.

Inflammatory markers in exhaled breath condensate in nonasthmatic children with food allergy

Allergy is a systemic inflammation; therefore, although the allergic symptom may be seen in a specific organ system, the effects of this inflammation may be seen in other organs. interleukin (IL) IL4, IL5 and IL13 are the major Th2 cytokines and e-cadherin is an epithelial barrier protein. The objective of this research was to assess indicators of inflammation specific to Th2 responses and proteins related to the protective barrier of the airway’s inner lining. These assessments were conducted using exhaled breath condensate (EBC), which provides insights into peripheral airway conditions of children suffering from food allergies. The study had 24 patients with food allergy and 24 control individuals younger than three years of age with no history of food reaction. The diagnosis of food allergy was based on food allergen-specific IgE and skin prick test positivity in our clinic and oral food testing in selected cases. EBC samples were obtained by Ecoscreen (Jaegar, Hoechberg, Germany). IL4, IL5, IL13 and E-cadherin levels were measured in these samples by enzyme linked immunoassay. The group of children with food allergies, consisting mainly of 14 girls, had a median age of 16 months, whereas the control group, which included 11 girls, had a median age of 15 months (p = 0.89). Comparing the two groups, children with food allergies exhibited notably lower levels of IL-13 in the EBC compared to the control group (median values of 59.14 and 76.36, respectively, p = 0.02). Conversely, the concentration of IL-4 in the EBC was significantly higher in children with food allergies (median values of 1.94 and 1.29, respectively, p = 0.003). However, the levels of IL-5 and e-cadherin showed no significant differences between the two groups (with p-values of 0.74 and 0.09, respectively) as shown in table . High level of IL-4 despite the low level of IL-13 in the EBC of children having food allergy may be indicative of an early inflammatory phase that is not yet in the effector phase. Studies about the evolution of this process later in life are needed to assess the role of airway inflammation in children with food allergy who develop asthma.

Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice.

Bone fractures are among the most prevalent musculoskeletal injuries, and pain management is an essential part of fracture treatment. Fractures heal through an early inflammatory phase, followed by repair and remodeling. Nonsteroidal anti-inflammatory drugs (NSAIDs) are not recommended for fracture pain control as they potently inhibit the inflammatory phase and, thus, impair the healing. Opioids do not provide a better alternative for several reasons, including abuse potential. Accordingly, there is an unmet clinical need for analgesics that effectively ameliorate postfracture pain without impeding the healing. Here, we investigated the analgesic efficacy of two nonpsychotropic cannabinoids, cannabidiol (CBD) and cannabigerol (CBG), in a mouse model for tibial fracture. Mice with fractured tibiae exhibited increased sensitivity to mechanical, cold, and hot stimuli. Both CBD and CBG normalized pain sensitivity to all tested stimuli, and their analgesic effects were comparable to those of the NSAIDs. Interestingly, CBD and CBG promoted bone healing via multiple mechanisms during the early and late phases. During the early inflammatory phase, both cannabinoids increased the abundance of periosteal bone progenitors in the healing hematoma and promoted the osteogenic commitment of these progenitors. During the later phases of healing, CBD and CBG accelerated the fibrocartilaginous callus mineralization and enhanced the viability and proliferation of bone and bone-marrow cells. These effects culminated in higher bone volume fraction, higher bone mineral density, and improved mechanical quality of the newly formed bone. Together, our data suggest CBD and CBG as therapeutic agents that can replace NSAIDs in managing postfracture pain as both cannabinoids exert potent analgesic effects and, at the same time, promote bone healing. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

The anti-inflammatory effects of mesenchymal stem cells attenuate diffuse pulmonary hemorrhage.

There are few effective treatment options for diffuse pulmonary hemorrhage (DPH). We aimed to elucidate the therapeutic role and underlying mechanisms of mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (MSC-EVs) in DPH. Therapeutic effects of MSCs/MSC-EVs in pristane-induced DPH mice were evaluated via pulmonary function testing and histopathology. Transcriptome sequencing analyzed differentially expressed genes in control, DPH, and MSC groups. The proportion of macrophage polarization was evaluated in vivo and in vitro via fluorescence-activated cell sorting in control, DPH, MSC, MSC-EV inhalation, and MSC-EV intravenous groups. Intraperitoneal injection of pristane induced diffuse alveolar hemorrhage, early fibrosis, and inflammation in C57BL/6 mice. Monocytes were depleted in the peripheral blood in DPH mice and MSCs were recruited to the lungs, resulting in significantly attenuated diffuse alveolar hemorrhage and suppressed immunological response. This was more effective in the hyperacute hemorrhage phase than the early inflammatory phase. An MSC treatment-mediated anti-inflammatory effect was observed in DPH mice. Furthermore, MSC-EVs inhalation or tail-vein injection could effectively reduce DPH injury. MSCs could suppress macrophage M1 polarization in DPH in vivo and in vitro. MSCs displayed significant therapeutic effects in pristane-induced DPH, which may be a promising cell-free therapeutic approach.

Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity.

Bone fractures, the most common musculoskeletal injuries, heal through three main phases: inflammatory, repair, and remodeling. Around 10% of fracture patients suffer from impaired healing that requires surgical intervention, a huge burden on the healthcare system. The rate of impaired healing increases with metabolic diseases such as obesity-associated hyperglycemia/type 2 diabetes (T2D), an increasing concern given the growing incidence of obesity/T2D. Immune cells play pivotal roles in fracture healing, and obesity/T2D is associated with defective immune-cell functions. However, there is a gap in knowledge regarding the stoichiometry of immune cells that populate the callus and how that population changes during different phases of healing. Here, we used complementary global and single-cell techniques to characterize the repertoire of immune cells in the fracture callus and to identify populations specifically enriched in the fracture callus relative to the unfractured bone or bone marrow. Our analyses identified two clear waves of immune-cell infiltration into the callus: the first wave occurs during the early inflammatory phase of fracture healing, while the second takes place during the late repair/early remodeling phase, which is consistent with previous publications. Comprehensive analysis of each wave revealed that innate immune cells were activated during the early inflammatory phase, but in later phases they returned to homeostatic numbers and activation levels. Of the innate immune cells, distinct subsets of activated dendritic cells were particularly enriched in the inflammatory healing hematoma. In contrast to innate cells, lymphocytes, including B and T cells, were enriched and activated in the callus primarily during the late repair phase. The Diet-Induced Obesity (DIO) mouse, an established model of obesity-associated hyperglycemia and insulin resistance, suffers from multiple healing defects. Our data demonstrate that DIO mice exhibit dysregulated innate immune responses during the inflammatory phase, and defects in all lymphocyte compartments during the late repair phase. Taken together, our data characterize, for the first time, immune populations that are enriched/activated in the callus during two distinct phases of fracture healing and identify defects in the healing-associated immune response in DIO mice, which will facilitate future development of immunomodulatory therapeutics for impaired fracture healing.

HMGB1 released by mesothelial cells drives the development of asbestos-induced mesothelioma

Asbestos is the main cause of malignant mesothelioma. Previous studies have linked asbestos-induced mesothelioma to the release of HMGB1 from the nucleus to the cytoplasm, and from the cytoplasm to the extracellular space. In the cytoplasm, HMGB1 induces autophagy impairing asbestos-induced cell death. Extracellularly, HMGB1 stimulates the secretion of TNFα. Jointly, these two cytokines kick-start a chronic inflammatory process that over time promotes mesothelioma development. Whether the main source of extracellular HMGB1 were the mesothelial cells, the inflammatory cells, or both was unsolved. This information is critical to identify the targets and design preventive/therapeutic strategies to interfere with asbestos-induced mesothelioma. To address this issue, we developed the conditional mesothelial HMGB1-knockout (Hmgb1ΔpMeso) and the conditional myelomonocytic-lineage HMGB1-knockout (Hmgb1ΔMylc) mouse models. We establish here that HMGB1 is mainly produced and released by the mesothelial cells during the early phases of inflammation following asbestos exposure. The release of HMGB1 from mesothelial cells leads to atypical mesothelial hyperplasia, and in some animals, this evolves over the years into mesothelioma. We found that Hmgb1ΔpMeso, whose mesothelial cells cannot produce HMGB1, show a greatly reduced inflammatory response to asbestos, and their mesothelial cells express and secrete significantly reduced levels of TNFα. Moreover, the tissue microenvironment in areas of asbestos deposits displays an increased fraction of M1-polarized macrophages compared to M2 macrophages. Supporting the biological significance of these findings, Hmgb1ΔpMeso mice showed a delayed and reduced incidence of mesothelioma and an increased mesothelioma-specific survival. Altogether, our study provides a biological explanation for HMGB1 as a driver of asbestos-induced mesothelioma.

Hydrolyzed Collagen Powder Dressing Improves Wound Inflammation, Perfusion, and Breaking Strength of Repaired Tissue.

Objective: Hydrolyzed collagen-based matrices are widely used as wound care dressings. Information on the mechanism of action of such dressings is scanty. The objective of this study was to test the effect of a specific hydrolyzed collagen powder (HCP), which is extensively used for wound care management in the United States. Approach: The effects of HCP on resolution of wound inflammation, perfusion, closure, and breaking strength of the repaired skin were studied in an experimental murine model. Results: In early (day 7) inflammatory phase of wound macrophages, HCP treatment boosted phagocytosis and efferocytosis of wound-site macrophages. In these cells, inducible reactive oxygen species were also higher on day (d) 7. HCP treatment potentiated the expression of anti-inflammatory interleukin (IL)-10 cytokine and proangiogenic vascular endothelial growth factor (VEGF) production. Excisional wounds dressed with HCP showed complete closure on day 21, while the control wounds remained open. HCP treatment also demonstrated improved quality of wound healing as marked by the improved breaking strength of the closed wound tissue/repaired skin. Innovation: These data represent first evidence on the mechanism of action of clinically used HCP. Conclusion: HCP dressing favorably influenced both wound inflammation and vascularization. Improved breaking strength of HCP-treated repaired skin lays the rationale for future studies testing the hypothesis that HCP-treated closed wounds would show fewer recurrences.

Analysis of In Vitro Leukocyte Responses to Biomaterials in the Presence of Antimicrobial Porcine Neutrophil Extract (AMPNE).

Implant insertion can evoke excessive inflammation which disrupts the healing process and potentially leads to complications such as implant rejection. Neutrophils and macrophages play a vital role in the early inflammatory phase of tissue repair, necessitating the study of cellular responses in host-implant interactions. In order to deepen the knowledge about these interactions, the response of neutrophils and macrophages to contact with selected biomaterials was examined in vitro on the basis of secretory response as well as reactive oxygen species/reactive nitrogen species (ROS/RNS) generation. Porcine neutrophils exposed to hydroxyapatite (HA) released more enzymes and generated higher levels of ROS/RNS compared to the control group. The addition of AMPNE diminished these responses. Although the results from porcine cells can provide valuable preliminary data, further validation using human cells or clinical studies would be necessary to fully extrapolate the findings to human medicine. Our study revealed that human neutrophils after contact of with HA increased the production of nitric oxide (NO) (10.00 ± 0.08 vs. control group 3.0 ± 0.11 µM, p < 0.05), while HAP or FAP did not elicit a significant response. Human macrophages cultured with HA produced more superoxide and NO, while HAP or FAP had a minimal effect, and curdlan reduced ROS/RNS generation. The addition of AMPNE to cultures with all biomaterials, except curdlan, reduced neutrophil activity, regardless of the peptides' origin. These results highlight the potential of antimicrobial peptides in modulating excessive biomaterial/host cell reactions involving neutrophils and macrophages, enhancing our understanding of immune reactions, and suggesting that AMPNE could regulate leukocyte response during implantation.

Effect of Adipose Derived Stromal Vascular Fraction on The Early Inflammatory Phase of Burn Wound Healing in Male Rat Model

Objectives: Severe burn treatment remains challenging. Research has investigated the efficacy of stem cells and stromal vascular fraction (SVF) in many types of injuries. SVF has advantages for its heterogenous population of cells and avoidance of culturing and ethical issues. SVF can be isolated either enzymatically or mechanically. Enzymatically isolated SVF reduced inflammation and enhanced neovascularization and re-epithelization in the treatment of burn injury. This study highlighted the efficacy of applying mechanically isolated SVF on the early inflammatory phase of deep partial-thickness burn by investigating toll-like receptor 4 (TLR4) signaling pathway.&#x0D; Methods: 30 male Wistar rats with deep partial-thickness burns were assigned into: the control group, silver sulfadiazine (SSD) cream group, and mechanically isolated SVF group. Morphological, histopathological evaluation of inflammation, and immunohistochemical analysis for (TLR4, TNF-α, IL‐1β, and IL-6) were evaluated for the first eight days post-treatment.&#x0D; Results: Morphologically, the SVF group significantly reduced edema and increased wound bed dryness on day one compared to the control group (P=0.0001) and to the SSD group (P=0.001). Histopathologically, the SVF group significantly reduced inflammation four days post-treatment compared with the control group (P=0.045). The SVF group significantly reduced TLR4 protein expression on days four and eight post-treatment compared with the control group (P= 0.001, P= 0.042, respectively). The TNF-α protein expression in the SVF group was significantly lower on days four and eight post-treatment compared with the control group (P= 0.046, P= 0.046, respectively) and with the SSD group (P= 0.008, P= 0.001, respectively). The IL‐1β expression was significantly reduced in the SVF group compared to the control group on day four post-treatment (P= 0.017). There were no significant differences in IL-6 expressions between all groups on both days.&#x0D; Conclusion: The mechanical isolation of SVF has an early anti-inflammatory impact on deep partial-thickness burn injury. This effect could be through inhibiting TLR4, TNF-α, and IL‐1β pathways. This could partially explain the mechanism behind SVF efficacy in the healing process of burn injury.