CTGF promotes the repair and regeneration of alveoli after acute lung injury by promoting the proliferation of subpopulation of AEC2s.

BACKGROUND: Functional alveolar regeneration is essential for the restoration of normal lung homeostasis after acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Lung is a relatively quiescent organ and a variety of stem cells are recruited to participate in lung repair and regeneration after lung tissue injury. However, there is still no effective method for promoting the proliferation of endogenous lung stem cells to promote repair and regeneration. METHODS: Using protein mass spectrometry analysis, we analyzed the microenvironment after acute lung injury. RNA sequencing and image cytometry were used in the alveolar epithelial type 2 cells (AEC2s) subgroup identification. Then we used Sftpc+AEC2 lineage tracking mice and purified AEC2s to further elucidate the molecular mechanism by which CTGF regulates AEC2s proliferation both in vitro and in vivo. Bronchoalveolar lavage fluid (BALF) from thirty ARDS patients who underwent bronchoalveolar lavage was collected for the analysis of the correlation between the expressing of Krt5 in BALF and patients' prognosis. RESULTS: Here, we elucidate that AEC2s are the main facultative stem cells of the distal lung after ALI and ARDS. The increase of connective tissue growth factor (CTGF) in the microenvironment after ALI promoted the proliferation of AEC2s subpopulations. Proliferated AEC2s rapidly expanded and differentiated into alveolar epithelial type 1 cells (AEC1s) in the regeneration after ALI. CTGF initiates the phosphorylation of LRP6 by promoting the interaction between Krt5 and LRP6 of AEC2s, thus activating the Wnt signaling pathway, which is the molecular mechanism of CTGF promoting the proliferation of AEC2s subpopulation. CONCLUSIONS: Our study verifies that CTGF promotes the repair and regeneration of alveoli after acute lung injury by promoting the proliferation of AEC2s subpopulation.

A New Method for Programmable RNA Editing Using CRISPR Effector Cas13X.1.

Type VI CRISPR-Cas13 is the only CRISPR system that can bind and cleave RNA without DNase activity. We used the newly discovered, smaller Cas13X.1 protein to construct an editing system in mammalian cells, aiming to break the delivery restrictions of CRISPR-Cas13 system in vivo and promote the application of Cas13X system in clinical therapy. We employed exogenous fluorescence reporter gene mCherry and endogenous gene transketolase (TKT) closely related to cancer cell metabolism as target genes to evaluate the Cas13X.1 system. The recombinant plasmids targeting exogenous gene mCherry and endogenous gene TKT were constructed based on Cas13X.1 backbone plasmid. The editing efficiency, protein expression level, downstream gene transcript level and safety of Cas13X.1 system were evaluated. Both TKT transcripts of endogenous genes and mCherry transcripts of exogenous genes were significantly degraded by Cas13X.1 system with a knockdown efficiency up to 50%. At the same time, Cas13X.1 down-regulated the expression of the corresponding protein level in the editing of transcripts. In addition, the transcripts of key metabolic enzymes related to TKT were also down-regulated synchronously, suggesting that the degradation of TKT transcripts by Cas13X.1 system affected the main metabolic pathways related to TKT. The morphology, RNA integrity and apoptosis of cells loaded with Cas13X.1 system were not affected. The Cas13X.1 system we constructed had strong RNA knockdown ability in mammalian cells with low cellular toxicity. Compared with other CRISPR-Cas13 systems, Cas13X.1 system with smaller molecular weight has more advantages in vivo delivery. The Cas13X.1 system targeting TKT transcripts also provides an alternative method for the study of anti-cancer therapy.

Endothelial-mesenchymal transition as a novel mechanism for generating myofibroblasts during wound healing and scarring.

BACKGROUND: The endothelial-mesenchymal transition (EndMT) is an important mechanism in tissue regeneration and the development of organ fibrosis. Whether EndMT occurs in wound healing and scarring remains unknown. MATERIALS AND METHODS: The isolated cells from the normal dermal tissue and the wound tissue of mouse with full-thickness skin wound, and human scar tissue sections were performed with CD31/factorVII and α-SMA immunohistochemical staining and H and E staining. The ratio of factor VII or CD31/α-SMA double-positive cells in factor VII-positive cells was assessed in the isolated cells and in scar tissues. RESULTS: In this study, we found that approximately 27-60% of ECs coexpressed VII factor and α-SMA in the isolated cells from the wound tissues of mice, which was significantly higher than that of normal dermal tissue cells. Accordingly, the number of CD31/α-SMA double-positive cells in mouse wound tissue sections was also significantly more than that in normal dermal tissue sections. In scar tissues, in addition to high-density microvessels, a large number of proliferative ECs in scar strama and CD31/α-SMA double-positive cells were also found. Approximately 46.82 to 84.11% of ECs and 68.77 to 95.25% of myofibroblasts coexpressed VII factor and α-SMA, and these two values in hypertrophic scars were significantly higher than those in keloids. CONCLUSION: These results confirmed that ECs might contribute to the emergence of myofibroblasts in the wound and scar tissue via the process of EndMT.

Sepsis-induced immunosuppression: mechanisms, diagnosis and current treatment options.

Sepsis is a common complication of combat injuries and trauma, and is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. It is also one of the significant causes of death and increased health care costs in modern intensive care units. The use of antibiotics, fluid resuscitation, and organ support therapy have limited prognostic impact in patients with sepsis. Although its pathophysiology remains elusive, immunosuppression is now recognized as one of the major causes of septic death. Sepsis-induced immunosuppression is resulted from disruption of immune homeostasis. It is characterized by the release of anti-inflammatory cytokines, abnormal death of immune effector cells, hyperproliferation of immune suppressor cells, and expression of immune checkpoints. By targeting immunosuppression, especially with immune checkpoint inhibitors, preclinical studies have demonstrated the reversal of immunocyte dysfunctions and established host resistance. Here, we comprehensively discuss recent findings on the mechanisms, regulation and biomarkers of sepsis-induced immunosuppression and highlight their implications for developing effective strategies to treat patients with septic shock.

PDGFR kinase inhibitor protects against septic death via regulation of BTLA.

Sepsis, defined as life-threatening organ failure caused by a dysregulated host response to severe infection, is a major cause of death among intensive care unit patients. Therapies targeting on immunomodulatory is a new research field in sepsis treatment. B- and T-lymphocyte attenuator (BTLA) is an inhibitory costimulatory factor molecule of B and T lymphocytes. Studies have shown that elevated expression of BTLA in lymphocytes can reduce mortality in sepsis, but its regulatory compounds and the underlying mechanism remains to be elucidated. Here, we show that treatment with CP-673451 significantly decreases mortality of septic mouse. CP-673451 is a PDGFR kinase inhibitor which can promote the expression of BTLA, inhibit the release of chemokines such as CXCL13, and reduce first the chemotaxis of B cells to the peripheral blood and vital organs. CP-673451 also inhibits both the release of cytokines and chemokines such as IL-1ß, IL-6, IL-10, TNF-α, CCL1, CCL2 and CCL7 and reduces both the chemotactic ability of T cells. This suggests that CP-673451 may prevent septic death by inhibiting lymphocyte chemotaxis and alleviating "cytokine storm". In conclusion, our study provides a new therapeutic target and an effective compound for sepsis treatment.

Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting.

PURPOSE: The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue. METHODS: Sprague-Dawley rats (3-4 weeks, 120-150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry. RESULTS: The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12-16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days. CONCLUSION: AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.

Tim-3 regulates sepsis-induced immunosuppression by inhibiting the NF-κB signaling pathway in CD4 T cells.

Immunosuppression in response to severe sepsis remains a serious human health concern. Evidence of sepsis-induced immunosuppression includes impaired T lymphocyte function, T lymphocyte depletion or exhaustion, increased susceptibility to opportunistic nosocomial infection, and imbalanced cytokine secretion. CD4 T cells play a critical role in cellular and humoral immune responses during sepsis. Here, using an RNA sequencing assay, we found that the expression of T cell-containing immunoglobulin and mucin domain-3 (Tim-3) on CD4 T cells in sepsis-induced immunosuppression patients was significantly elevated. Furthermore, the percentage of Tim-3+ CD4 T cells from sepsis patients was correlated with the mortality of sepsis-induced immunosuppression. Conditional deletion of Tim-3 in CD4 T cells and systemic Tim-3 deletion both reduced mortality in response to sepsis in mice by preserving organ function. Tim-3+ CD4 T cells exhibited reduced proliferative ability and elevated expression of inhibitory markers compared with Tim-3-CD4 T cells. Colocalization analyses indicated that HMGB1 was a ligand that binds to Tim-3 on CD4 T cells and that its binding inhibited the NF-κB signaling pathway in Tim-3+ CD4 T cells during sepsis-induced immunosuppression. Together, our findings reveal the mechanism of Tim-3 in regulating sepsis-induced immunosuppression and provide a novel therapeutic target for this condition.

A Functional Variant of CXCL16 Is Associated With Predisposition to Sepsis and MODS in Trauma Patients: Genetic Association Studies.

PURPOSE: CXC chemokines are mediators which mediate immune cells migration to sites of inflammation and injury. Chemokine C-X-C motif ligand 16 (CXCL16) plays an important role in the occurrence and development of sepsis through leukocyte chemotaxis, leukocyte adhesion and endotoxin clearance. In this study, we selected a set of tagging single nucleotide polymorphisms (tag SNPs) in the CXCL16 gene and investigated their clinical relevance to the development of sepsis and multiple organ dysfunction syndrome (MODS) in patients with major trauma in three independent Chinese Han populations. METHODS: A total of 1,620 major trauma patients were enrolled in this study. Among these patients, 920 came from Chongqing in western China, 350 came from Zhejiang Province in eastern China, and 350 came from Guizhou Province in southwestern China. The improved multiplex ligation detection reaction (iMLDR) method was employed in the genotyping and genetic association analyses to determine the associations between CXCL16 haplotypes and sepsis morbidity rate and higher MOD scores in three cohorts. RESULTS: Only CXCL16 T123V181 haplotype was associated with an increased risk for sepsis morbidity and higher MOD scores in the three cohorts (OR = 1.89, P = 0.001 for the Chongqing cohort; OR = 1.76, P = 0.004 for the Zhejiang cohort; OR = 1.55, P = 0.012 for the Guizhou cohort). The effect of T123V181 haplotype on the chemotaxis, migration and endotoxin clearance of immune cells were further observed. Protein modeling analysis showed that T123 and V181 might alter the structure of the CXCL16 active center. Thus it enhanced the chemotaxis and adhesion ability of immunocytes. CONCLUSION: We demonstrate the mechanism of CXCL16 T123V181 haplotype which regulates the sepsis morbidity rate and thus provide a new biomarker for early diagnosis of sepsis and MODS. CLINICAL TRIAL REGISTRATION: www.ClinicalTrials.gov, identifier NCT01713205 (https://www.clinicaltrials.gov/ct2/results?cond=&term=+NCT01713205&cntry=&state=&city=&dist=).

[Dynamic changes of cellular immune function in trauma patients and its relationship with prognosis].

OBJECTIVE: To study the dynamic changes of cellular immune function in peripheral blood of trauma patients and its role in the evaluation of traumatic complications. METHODS: A prospective cohort study design was conducted. Patients with blunt trauma admitted to Chongqing Emergency Medical Center from November 2019 to January 2020 were consecutively enrolled. The peripheral blood samples were collected at 1, 3, 5, 7, and 14 days after injury. The expressions of CD64, CD274, and CD279 on the surface of neutrophils, lymphocytes, and monocytes as well as CD3+, CD4+ and CD8+ T lymphocyte subsets were measured by flow cytometry. The trauma patients were divided into different groups according to the injury severity score (ISS) and sepsis within 28 days after injury, respectively. The dynamic changes of cellular immune function in different time points after injury and differences between different groups were compared. Furthermore, the correlation with acute physiology and chronic health evaluation II (APACHE II), sequential organ failure assessment (SOFA), and ISS were evaluated by Pearson correlation analysis. RESULTS: A total of 42 patients with trauma were finally enrolled, containing 8 severe trauma patients with ISS greater than 25 scores, 17 patients with ISS between 16 and 25 scores, and 17 patients with ISS less than 16 scores. The sepsis morbidity rates were 14.3% (n = 6) within 28 days after injury. CD64 index and CD4+ T lymphocyte subsets were significantly increased at different time points after trauma (H = 15.464, P = 0.004; F = 2.491, P = 0.035). The CD64 index and positive rates of CD279 in neutrophils, lymphocytes, and monocytes were increased with the severity of injury at day 1 and day 3 after injury, respectively. At the first day after injury, CD64 index were 2.81±1.79, 1.77±0.92, 3.49±1.09; positive rate of CD279 in neutrophils were 1.40% (0.32%, 2.04%), 0.95% (0.44%, 2.70%), 12.73% (3.00%, 25.20%); positive rate of CD279 in lymphocytes were 3.77% (3.04%, 5.15%), 4.71% (4.08%, 6.32%), 8.01% (4.59%, 11.59%); positive rate of CD279 in monocytes were 0.57% (0.24%, 1.09%), 0.85% (0.22%, 1.25%), 6.74% (2.61%, 18.94%) from mild to severe injury groups, respectively. The CD64 index in severe injury group was significantly higher than that in moderate group, and the positive rates of CD279 in neutrophils, lymphocytes and monocytes of severe injury patients were higher than those in other two groups (all P < 0.05). At 3rd day after injury, compared to moderate group, severe injury patients had significantly higher CD64 index and positive rate of CD279 in lymphocytes [4.58±2.41 vs. 2.43±1.68, 7.35% (5.90%, 12.28%) vs. 4.63% (3.26%, 6.06%), both P < 0.05]. Compared with the non-sepsis patients, the sepsis patients had significantly higher CD64 index and positive rate of CD279 in monocytes at day 1 after injury [4.06±1.72 vs. 2.36±1.31, 3.29% (1.14%, 12.84%) vs. 0.67% (0.25%, 1.48%), both P < 0.05], and positive rate of CD279 in lymphocytes significantly higher at 3rd day after injury [8.73% (7.52%, 15.82%) vs. 4.67% (3.82%, 6.21%), P < 0.05]. In addition, correlation analysis showed that positive rate of CD279 in lymphocytes was positively correlated with SOFA and ISS, respectively (r values were 0.533 and 0.394, both P < 0.05), positive rate of CD279 in monocytes was positively correlated with APACHE II, SOFA and ISS scores, respectively (r values were 0.579, 0.452 and 0.490, all P < 0.01), positive rate of CD279 in neutrophils was positively correlated with APACHE II and ISS, respectively (r values were 0.358 and 0.388, both P < 0.05). CONCLUSIONS: CD64 index and CD279 expression in neutrophils, lymphocytes, and monocytes are significantly related to the severity and prognosis of trauma. Dynamic monitoring the cellular immune function may be helpful for assessing the prognosis of trauma patients.