Complement receptor 3-dependent engagement by Candida glabrata ß-glucan modulates dendritic cells to induce regulatory T-cell expansion.

Candida glabrata is an important pathogen causing invasive infection associated with a high mortality rate. One mechanism that causes the failure of Candida eradication is an increase in regulatory T cells (Treg), which play a major role in immune suppression and promoting Candida pathogenicity. To date, how C. glabrata induces a Treg response remains unclear. Dendritic cells (DCs) recognition of fungi provides the fundamental signal determining the fate of the T-cell response. This study investigated the interplay between C. glabrata and DCs and its effect on Treg induction. We found that C. glabrata ß-glucan was a major component that interacted with DCs and consequently mediated the Treg response. Blocking the binding of C. glabrata ß-glucan to dectin-1 and complement receptor 3 (CR3) showed that CR3 activation in DCs was crucial for the induction of Treg. Furthermore, a ligand-receptor binding assay showed the preferential binding of C. glabrata ß-glucan to CR3. Our data suggest that C. glabrata ß-glucan potentially mediates the Treg response, probably through CR3-dependent activation in DCs. This study contributes new insights into immune modulation by C. glabrata that may lead to a better design of novel immunotherapeutic strategies for invasive C. glabrata infection.

Polymeric Particle BAM15 Targeting Macrophages Attenuates the Severity of LPS-Induced Sepsis: A Proof of Concept for Specific Immune Cell-Targeted Therapy.

Macrophage polarization requires different energy sources and metabolic processes. Therefore, cell energy interference to alter macrophage functions has been proposed as a treatment for severe inflammatory diseases, including sepsis. In this study, targeting cell energy using BAM15 (a mitochondrial uncoupling agent) in human THP-1 and mouse RAW264.7 macrophages prominently interfered with M1 but not M2 polarization. Free BAM15 (BAM15) and BAM15-loaded PLGA particles (BAM15 particles) reduced the inflammatory response of M1 macrophages and enhanced the expression of M2 signature genes with the restoration of mitochondrial activity (extracellular flux analysis) in RAW264.7 cells. Furthermore, BAM15 particles but not BAM15 showed specific effects on the inflammatory response of macrophages but not neutrophils, and the particles were actively captured by splenic and liver macrophages in vivo. Administration of BAM15 and BAM15 particles attenuated the severity of sepsis in LPS-induced sepsis mice. Interestingly, BAM15 particles but not BAM15 alleviated LPS-induced liver injury by reducing hepatic inflammation. Our findings substantiate the superior efficacy of macrophage-targeted therapy using a BAM15 particle-delivery system and provide further support for clinical development as a potential therapy for severe inflammatory diseases.

Immunosuppressive Polymeric Nanoparticles Targeting Dendritic Cells Alleviate Lupus Disease in Fcgr2b-/- Mice by Mediating Antigen-Specific Immune Tolerance.

Dendritic cells (DCs) are the most potent antigen-presenting cells that have multifaceted functions in the control of immune activation and tolerance. Hyperresponsiveness and altered tolerogenicity of DCs contribute to the development and pathogenesis of system lupus erythematosus (SLE); therefore, DC-targeted therapies aimed at inducing specific immune tolerance have become of great importance for the treatment of SLE. This study developed a new nanoparticle (NP) containing a biodegradable PDMAEMA-PLGA copolymer for target-oriented delivery to DCs in situ. PDMAEMA-PLGA NPs provided sustained drug release and exhibited immunosuppressive activity in FLT3L and GM-CSF-derived bone marrow in conventional DCs (BM-cDCs). PDMAEMA-PLGA NPs improved dexamethasone capability to convert wild-type and Fcgr2b-/- BM-cDCs from an immunogenic to tolerogenic state, and BM-cDCs treated with dexamethasone-incorporated PDMAEMA-PLGA NPs (Dex-NPs) efficiently mediated regulatory T cell (Treg) expansion in vitro. Dex-NP therapy potentially alleviated lupus disease in Fcgr2b-/- mice by mediating Foxp3+ Treg expansion in an antigen-specific manner. Our findings substantiate the superior efficacy of DC-targeted therapy using the PDMAEMA-PLGA NP delivery system and provide further support for clinical development as a potential therapy for SLE. Furthermore, PDMAEMA-PLGA NP may be a versatile platform for DC-targeted therapy to induce antigen-specific immune tolerance to unwanted immune responses that occur in autoimmune disease, allergy, and transplant rejection.

Lacticaseibacillus rhamnosus dfa1 Attenuate Cecal Ligation-Induced Systemic Inflammation through the Interference in Gut Dysbiosis, Leaky Gut, and Enterocytic Cell Energy.

Despite an uncommon condition, the clinical management of phlegmon appendicitis (retention of the intra-abdominal appendiceal abscess) is still controversial, and probiotics might be partly helpful. Then, the retained ligated cecal appendage (without gut obstruction) with or without oral Lacticaseibacillus rhamnosus dfa1 (started at 4 days prior to the surgery) was used as a representative model. At 5 days post-surgery, the cecal-ligated mice demonstrated weight loss, soft stool, gut barrier defect (leaky gut using FITC-dextran assay), fecal dysbiosis (increased Proteobacteria with reduced bacterial diversity), bacteremia, elevated serum cytokines, and spleen apoptosis without kidney and liver damage. Interestingly, the probiotics attenuated disease severity as indicated by stool consistency index, FITC-dextran assay, serum cytokines, spleen apoptosis, fecal microbiota analysis (reduced Proteobacteria), and mortality. Additionally, impacts of anti-inflammatory substances from culture media of the probiotics were demonstrated by attenuation of starvation injury in the Caco-2 enterocyte cell line as indicated by transepithelial electrical resistance (TEER), inflammatory markers (supernatant IL-8 with gene expression of TLR4 and NF-κB), cell energy status (extracellular flux analysis), and the reactive oxygen species (malondialdehyde). In conclusion, gut dysbiosis and leaky-gut-induced systemic inflammation might be helpful clinical parameters for patients with phlegmon appendicitis. Additionally, the leaky gut might be attenuated by some beneficial molecules from probiotics.

Blood Bacteria-Free DNA in Septic Mice Enhances LPS-Induced Inflammation in Mice through Macrophage Response.

Although bacteria-free DNA in blood during systemic infection is mainly derived from bacterial death, translocation of the DNA from the gut into the blood circulation (gut translocation) is also possible. Hence, several mouse models with experiments on macrophages were conducted to explore the sources, influences, and impacts of bacteria-free DNA in sepsis. First, bacteria-free DNA and bacteriome in blood were demonstrated in cecal ligation and puncture (CLP) sepsis mice. Second, administration of bacterial lysate (a source of bacterial DNA) in dextran sulfate solution (DSS)-induced mucositis mice elevated blood bacteria-free DNA without bacteremia supported gut translocation of free DNA. The absence of blood bacteria-free DNA in DSS mice without bacterial lysate implies an impact of the abundance of bacterial DNA in intestinal contents on the translocation of free DNA. Third, higher serum cytokines in mice after injection of combined bacterial DNA with lipopolysaccharide (LPS), when compared to LPS injection alone, supported an influence of blood bacteria-free DNA on systemic inflammation. The synergistic effects of free DNA and LPS on macrophage pro-inflammatory responses, as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10), pro-inflammatory genes (NFκB, iNOS, and IL-1ß), and profound energy alteration (enhanced glycolysis with reduced mitochondrial functions), which was neutralized by TLR-9 inhibition (chloroquine), were demonstrated. In conclusion, the presence of bacteria-free DNA in sepsis mice is partly due to gut translocation of bacteria-free DNA into the systemic circulation, which would enhance sepsis severity. Inhibition of the responses against bacterial DNA by TLR-9 inhibition could attenuate LPS-DNA synergy in macrophages and might help improve sepsis hyper-inflammation in some situations.

Interference on Cytosolic DNA Activation Attenuates Sepsis Severity: Experiments on Cyclic GMP-AMP Synthase (cGAS) Deficient Mice.

Although the enhanced responses against serum cell-free DNA (cfDNA) in cases of sepsis-a life-threatening organ dysfunction due to systemic infection-are understood, the influence of the cytosolic DNA receptor cGAS (cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase) on sepsis is still unclear. Here, experiments on cGAS deficient (cGAS-/-) mice were conducted using cecal ligation and puncture (CLP) and lipopolysaccharide (LPS) injection sepsis models and macrophages. Severity of CLP in cGAS-/- mice was less severe than in wildtype (WT) mice, as indicated by mortality, serum LPS, cfDNA, leukopenia, cytokines (TNF-α, IL-6 and IL-10), organ histology (lung, liver and kidney) and spleen apoptosis. With the LPS injection model, serum cytokines in cGAS-/- mice were lower than in WT mice, despite the similar serum cfDNA level. Likewise, in LPS-activated WT macrophages, the expression of several mitochondria-associated genes (as revealed by RNA sequencing analysis) and a profound reduction in mitochondrial parameters, including maximal respiration (determined by extracellular flux analysis), DNA (mtDNA) and mitochondrial abundance (revealed by fluorescent staining), were demonstrated. These data implied the impact of cfDNA resulting from LPS-induced cell injury. In parallel, an additive effect of bacterial DNA on LPS, seen in comparison with LPS alone, was demonstrated in WT macrophages, but not in cGAS-/- cells, as indicated by supernatant cytokines (TNF-α and IL-6), M1 proinflammatory polarization (iNOS and IL-1ß), cGAS, IFN-γ and supernatant cyclic GMP-AMP (cGAMP). In conclusion, cGAS activation by cfDNA from hosts (especially mtDNA) and bacteria was found to induce an additive proinflammatory effect on LPS-activated macrophages which was perhaps responsible for the more pronounced sepsis hyperinflammation observed in WT mice compared with the cGAS-/- group.