Caspase-11 interaction with NLRP3 potentiates the noncanonical activation of the NLRP3 inflammasome.

Caspase-11 detection of intracellular lipopolysaccharide (LPS) from invasive Gram-negative bacteria mediates noncanonical activation of the NLRP3 inflammasome. While avirulent bacteria do not invade the cytosol, their presence in tissues necessitates clearance and immune system mobilization. Despite sharing LPS, only live avirulent Gram-negative bacteria activate the NLRP3 inflammasome. Here, we found that bacterial mRNA, which signals bacterial viability, was required alongside LPS for noncanonical activation of the NLRP3 inflammasome in macrophages. Concurrent detection of bacterial RNA by NLRP3 and binding of LPS by pro-caspase-11 mediated a pro-caspase-11-NLRP3 interaction before caspase-11 activation and inflammasome assembly. LPS binding to pro-caspase-11 augmented bacterial mRNA-dependent assembly of the NLRP3 inflammasome, while bacterial viability and an assembled NLRP3 inflammasome were necessary for activation of LPS-bound pro-caspase-11. Thus, the pro-caspase-11-NLRP3 interaction nucleated a scaffold for their interdependent activation explaining their functional reciprocal exclusivity. Our findings inform new vaccine adjuvant combinations and sepsis therapy.

Secreted IgD Amplifies Humoral T Helper 2 Cell Responses by Binding Basophils via Galectin-9 and CD44.

B cells thwart antigenic aggressions by releasing immunoglobulin M (IgM), IgG, IgA, and IgE, which deploy well-understood effector functions. In contrast, the role of secreted IgD remains mysterious. We found that some B cells generated IgD-secreting plasma cells following early exposure to external soluble antigens such as food proteins. Secreted IgD targeted basophils by interacting with the CD44-binding protein galectin-9. When engaged by antigen, basophil-bound IgD increased basophil secretion of interleukin-4 (IL-4), IL-5, and IL-13, which facilitated the generation of T follicular helper type 2 cells expressing IL-4. These germinal center T cells enhanced IgG1 and IgE but not IgG2a and IgG2b responses to the antigen initially recognized by basophil-bound IgD. In addition, IgD ligation by antigen attenuated allergic basophil degranulation induced by IgE co-ligation. Thus, IgD may link B cells with basophils to optimize humoral T helper type 2-mediated immunity against common environmental soluble antigens.

B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen.

Neutrophils use immunoglobulins to clear antigen, but their role in immunoglobulin production is unknown. Here we identified neutrophils around the marginal zone (MZ) of the spleen, a B cell area specialized in T cell-independent immunoglobulin responses to circulating antigen. Neutrophils colonized peri-MZ areas after postnatal mucosal colonization by microbes and enhanced their B cell-helper function after receiving reprogramming signals, including interleukin 10 (IL-10), from splenic sinusoidal endothelial cells. Splenic neutrophils induced immunoglobulin class switching, somatic hypermutation and antibody production by activating MZ B cells through a mechanism that involved the cytokines BAFF, APRIL and IL-21. Neutropenic patients had fewer and hypomutated MZ B cells and a lower abundance of preimmune immunoglobulins to T cell-independent antigens, which indicates that neutrophils generate an innate layer of antimicrobial immunoglobulin defense by interacting with MZ B cells.

The transmembrane activator TACI triggers immunoglobulin class switching by activating B cells through the adaptor MyD88.

BAFF and APRIL are innate immune mediators that trigger immunoglobulin G (IgG) and IgA class-switch recombination (CSR) in B cells by engaging the receptor TACI. The mechanism that underlies CSR signaling by TACI remains unknown. Here we found that the cytoplasmic domain of TACI encompasses a conserved motif that bound MyD88, an adaptor that activates transcription factor NF-kappaB signaling pathways via a Toll-interleukin 1 (IL-1) receptor (TIR) domain. TACI lacks a TIR domain, yet triggered CSR via the DNA-editing enzyme AID by activating NF-kappaB through a Toll-like receptor (TLR)-like MyD88-IRAK1-IRAK4-TRAF6-TAK1 pathway. TACI-induced CSR was impaired in mice and humans lacking MyD88 or the kinase IRAK4, which indicates that MyD88 controls a B cell-intrinsic, TIR-independent, TACI-dependent pathway for immunoglobulin diversification.

HIV-1 evades virus-specific IgG2 and IgA responses by targeting systemic and intestinal B cells via long-range intercellular conduits.

Contact-dependent communication between immune cells generates protection but also facilitates viral spread. Here we found that macrophages formed long-range actin-propelled conduits in response to negative factor (Nef), a human immunodeficiency virus type 1 (HIV-1) protein with immunosuppressive functions. Conduits attenuated immunoglobulin G2 (IgG2) and IgA class switching in systemic and intestinal lymphoid follicles by shuttling Nef from infected macrophages to B cells through a guanine-exchange factor-dependent pathway involving the amino-terminal anchor, central core and carboxy-terminal flexible loop of Nef. By showing stronger virus-specific IgG2 and IgA responses in patients with Nef-deficient virions, our data suggest that HIV-1 exploits intercellular 'highways' as a 'Trojan horse' to deliver Nef to B cells and evade humoral immunity systemically and at mucosal sites of entry.

Polysaccharides from Polygonatum kingianum Collett & Hemsl ameliorated fatigue by regulating NRF2/HO-1/NQO1 and AMPK/PGC-1α/TFAM signaling pathways, and gut microbiota.

Polygonatum kingianum Coll & Hemsl is an important Chinese medicine used for enhancing physical function and anti-fatigue, and polysaccharides (PKPs) are considered as the main bioactive components. However, the mechanisms through which PKPs exert their anti-fatigue effects are not fully understood. This study aimed more comprehensively to explore the anti-fatigue mechanisms of PKPs, focusing on metabolism, protein expression, and gut flora, by using exhaustive swimming experiments in mice. Results showed a significant increase in the exhaustive swimming time of the mice treated with PKPs, especially in the high-dose group (200 mg/kg/day). Further studies showed that PKPs remarkably improves several fatigue-related physiological indices. Additionally, 16S rRNA sequence analysis showed that PKPs increased antioxidant bacteria (e.g., g_norank_f_Muribaculaceae) and the production of short-chain fatty acids (SCFAs), while reducing the abundance of harmful bacteria (e.g., g_Escherichia-Shigella and g_Helicobacter). PKPs also mitigated oxidative stress through activating the NRF2/HO-1 signaling pathway, and promoted energy metabolism by upregulating the expression of AMPK/PGC-1α/TFAM signaling pathway proteins. This research may offer theoretical support for incorporating PKPs as a novel dietary supplement in functional foods targeting anti-fatigue properties.

The Protective Effect and Mechanism of a Phytochemical Extract from the Wild Vegetable Shutou (Crateva unilocularis Buch.) against Acetaminophen-Induced Liver Injury in Mice.

Acetaminophen (APAP) abuse is a common public health problem which can cause severe liver damage. However, strategies for dealing with this situation safely and effectively are very limited. The goal of the current work was to evaluate the protection and potential molecular mechanisms of an ethanol extract from shoots of the wild vegetable shutou (Crateva unilocularis Buch.) (ECS) against APAP-induced liver damage in mice. Mice orally received ECS for seven days (300 or 600 mg/kg b.w. per day) before being intraperitoneally injected with APAP (250 mg/kg). Results exhibited that ECS obviously decreased the content of alkaline phosphatase, alanine aminotransferase, aspartate transaminase, and malondialdehyde (p < 0.05). Catalase and superoxide dismutase were notably restored (p < 0.05), and the content of reduced glutathione was obviously increased (p < 0.05). Moreover, ECS significantly inhibited the secretion of interleukin-1ß and tumor necrosis factor-α (p < 0.05). Further analyses of the mechanisms showed that ECS may alleviate oxidative stress in the liver by increasing the expression of the nuclear factor erythroid-2-related factor 2 and NADH quinone oxidoreductase 1 proteins, and may suppress liver inflammation by inhibiting the expression of the phosphorylated-inhibitor kappa B alpha/inhibitor kappa B alpha, phosphorylated-nuclear factor κB/nuclear factor κB, and cyclooxygenase-2 proteins. Meanwhile, ECS inhibited hepatocyte apoptosis by enhancing B-cell lymphoma gene 2 and suppressing Bcl-2-associated X protein. In summary, ECS may be used as a dietary supplement to prevent the liver damage caused by APAP abuse.

The Efficacy of Rehabilitation Nursing Interventions on Patients with Open Lower Limb Fractures.

Objective: The study aims to analyze the efficacy of rehabilitation nursing interventions on patients with open lower limb fractures. Methods: From June 2020, patients who received RNI (observation group) were included and compared with patients who received routine nursing interventions (control group). The efficacy of different nursing modes was compared with several indicators. Results: One hundred patients were included in this study, 50 in each group. The baseline characteristics were not significantly different between the groups. Regarding the emotional scores, the Self-Rating Anxiety Scale (SAS) score (26.98 vs 43.47), and Distress Management Screening Measure (DMSM) score (8.01 vs 12.85) in the observation group were significantly lower than those in the control group, both P < 0.05. Regarding the postoperative related indexes, the postoperative pain score (10.13 vs 15.53), fracture healing time (6.32 vs 10.86 weeks), and postoperative complications rate (0 vs 12%) in the observation group were all significantly lower than those in the control group, all P < 0.05. Regarding the quality of life scores, the WHOQOL-100 score (94.12 vs 83.13) and PSQI score (6.43 vs 10.36) were both significantly better in the observation group, with both P < 0.05. Conclusion: Patients with open lower limbs who received RNI can help patients reduce postoperative anxiety and stress, promote postoperative rehabilitation and improve their quality of life.

HIV-1 gp120 mannoses induce immunosuppressive responses from dendritic cells.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a vaccine immunogen that can signal via several cell surface receptors. To investigate whether receptor biology could influence immune responses to gp120, we studied its interaction with human, monocyte-derived dendritic cells (MDDCs) in vitro. Gp120 from the HIV-1 strain JR-FL induced IL-10 expression in MDDCs from 62% of donors, via a mannose C-type lectin receptor(s) (MCLR). Gp120 from the strain LAI was also an IL-10 inducer, but gp120 from the strain KNH1144 was not. The mannose-binding protein cyanovirin-N, the 2G12 mAb to a mannose-dependent gp120 epitope, and MCLR-specific mAbs inhibited IL-10 expression, as did enzymatic removal of gp120 mannose moieties, whereas inhibitors of signaling via CD4, CCR5, or CXCR4 were ineffective. Gp120-stimulated IL-10 production correlated with DC-SIGN expression on the cells, and involved the ERK signaling pathway. Gp120-treated MDDCs also responded poorly to maturation stimuli by up-regulating activation markers inefficiently and stimulating allogeneic T cell proliferation only weakly. These adverse reactions to gp120 were MCLR-dependent but independent of IL-10 production. Since such mechanisms might suppress immune responses to Env-containing vaccines, demannosylation may be a way to improve the immunogenicity of gp120 or gp140 proteins.

Microbiota regulate the ability of lung dendritic cells to induce IgA class-switch recombination and generate protective gastrointestinal immune responses.

Protective immunoglobulin A (IgA) responses to oral antigens are usually orchestrated by gut dendritic cells (DCs). Here, we show that lung CD103(+) and CD24(+)CD11b(+) DCs induced IgA class-switch recombination (CSR) by activating B cells through T cell-dependent or -independent pathways. Compared with lung DCs (LDC), lung CD64(+) macrophages had decreased expression of B cell activation genes and induced significantly less IgA production. Microbial stimuli, acting through Toll-like receptors, induced transforming growth factor-ß (TGF-ß) production by LDCs and exerted a profound influence on LDC-mediated IgA CSR. After intranasal immunization with inactive cholera toxin (CT), LDCs stimulated retinoic acid-dependent up-regulation of α4ß7 and CCR9 gut-homing receptors on local IgA-expressing B cells. Migration of these B cells to the gut resulted in IgA-mediated protection against an oral challenge with active CT. However, in germ-free mice, the levels of LDC-induced, CT-specific IgA in the gut are significantly reduced. Herein, we demonstrate an unexpected role of the microbiota in modulating the protective efficacy of intranasal vaccination through their effect on the IgA class-switching function of LDCs.

The soluble pattern recognition receptor PTX3 links humoral innate and adaptive immune responses by helping marginal zone B cells.

Pentraxin 3 (PTX3) is a fluid-phase pattern recognition receptor of the humoral innate immune system with ancestral antibody-like properties but unknown antibody-inducing function. In this study, we found binding of PTX3 to splenic marginal zone (MZ) B cells, an innate-like subset of antibody-producing lymphocytes strategically positioned at the interface between the circulation and the adaptive immune system. PTX3 was released by a subset of neutrophils that surrounded the splenic MZ and expressed an immune activation-related gene signature distinct from that of circulating neutrophils. Binding of PTX3 promoted homeostatic production of IgM and class-switched IgG antibodies to microbial capsular polysaccharides, which decreased in PTX3-deficient mice and humans. In addition, PTX3 increased IgM and IgG production after infection with blood-borne encapsulated bacteria or immunization with bacterial carbohydrates. This immunogenic effect stemmed from the activation of MZ B cells through a neutrophil-regulated pathway that elicited class switching and plasmablast expansion via a combination of T cell-independent and T cell-dependent signals. Thus, PTX3 may bridge the humoral arms of the innate and adaptive immune systems by serving as an endogenous adjuvant for MZ B cells. This property could be harnessed to develop more effective vaccines against encapsulated pathogens.

DNA immune responses induced by codelivery of IL-12 expression vectors with hepatitis C structural antigens.

OBJECTIVE: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased immune responses. METHODS: Four recombinant plasmids constructed included the coding regions for the core protein (pC) and for the core, E1 and E2 together (pCE1E2), IL-12 p35 and p40. These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/C mice for measurement of specific antibodies and cytotoxic T-lymphocyte (CTL) responses. RESULTS: All the recombinant plasmids were shown to express specific antigens stably in mammalian cells. Codelivery of pIL-12 expression cassettes with pC and pCE1E2 in mice resulted in the enhancement of Ag-dependent CTL responses and the reduction of specific Ab response. The CTL activity was: pC=18.65%+/-5.71%, pCE1E2=20.07%+/-11.11%, pC+pIL-12=60.11%+/-17.37%, pCE1E2+pIL-12=67.48%+/-15.57%, respectively. The average A values of anti-HCV were pC=0.415+/-0.127, pCE1E2=0.358+/-0.096, pC+pIL-12=0.210+/-0.086, pCE1E2+pIL-12=0.258+/-0.125. CONCLUSION: Codelivery of pIL-12 with plasmid DNA can enhance the efficacy of immune responses and shift the type of immune responses.

Mucus enhances gut homeostasis and oral tolerance by delivering immunoregulatory signals.

A dense mucus layer in the large intestine prevents inflammation by shielding the underlying epithelium from luminal bacteria and food antigens. This mucus barrier is organized around the hyperglycosylated mucin MUC2. Here we show that the small intestine has a porous mucus layer, which permitted the uptake of MUC2 by antigen-sampling dendritic cells (DCs). Glycans associated with MUC2 imprinted DCs with anti-inflammatory properties by assembling a galectin-3-Dectin-1-FcγRIIB receptor complex that activated ß-catenin. This transcription factor interfered with DC expression of inflammatory but not tolerogenic cytokines by inhibiting gene transcription through nuclear factor κB. MUC2 induced additional conditioning signals in intestinal epithelial cells. Thus, mucus does not merely form a nonspecific physical barrier, but also constrains the immunogenicity of gut antigens by delivering tolerogenic signals.

Epithelial cells trigger frontline immunoglobulin class switching through a pathway regulated by the inhibitor SLPI.

Epithelial cells (ECs) transport class-switched immunoglobulin G (IgG) and IgA antibodies across mucous membranes. Whether ECs initiate class switching remains unknown. Here we found that ECs lining tonsillar crypts formed pockets populated by B cells expressing activation-induced cytidine deaminase (AID), an enzyme associated with ongoing class switching. ECs released B cell-activating AID-inducing factors after sensing microbial products through Toll-like receptors. The resulting class switching was amplified by thymic stromal lymphopoietin, an epithelial interleukin 7-like cytokine that enhanced the B cell 'licensing' function of dendritic cells, and was restrained by secretory leukocyte protease inhibitor, an epithelial homeostatic protein that inhibited AID induction in B cells. Thus, ECs may function as mucosal 'guardians' orchestrating frontline IgG and IgA class switching through a Toll-like receptor-inducible signaling program regulated by secretory leukocyte protease inhibitor.NOTE: In the version of this article initially published online, the middle label above Figure 6c is incorrect. The correct label should be 'BAFF'. The error has been corrected for all versions of the article.

Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL.

Bacteria colonize the intestine shortly after birth and thereafter exert several beneficial functions, including induction of protective immunoglobulin A (IgA) antibodies. The distal intestine contains IgA(2), which is more resistant to bacterial proteases than is IgA(1). The mechanism by which B cells switch from IgM to IgA(2) remains unknown. We found that human intestinal epithelial cells (IECs) triggered IgA(2) class switching in B cells, including IgA(1)-expressing B cells arriving from mucosal follicles, through a CD4(+) T cell-independent pathway involving a proliferation-inducing ligand (APRIL). IECs released APRIL after sensing bacteria through Toll-like receptors (TLRs) and further increased APRIL production by activating dendritic cells via thymic stromal lymphopoietin. Our data indicate that bacteria elicit IgA(2) class switching by linking lamina propria B cells with IECs through a TLR-inducible signaling program requiring APRIL. Thus, mucosal vaccines should activate IECs to induce more effective IgA(2) responses.