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RNA-based therapeutics hold great promise for treating diseases and lipid nanoparticles (LNPs) represent the most advanced platform for RNA delivery. However, the fate of the LNP-mRNA after endosome-engulfing and escape from the autophagy-lysosomal pathway remains unclear. To investigate this, mRNA (encoding human erythropoietin) was delivered to cells using LNPs, which shows, for the first time, a link between LNP-mRNA endocytosis and its packaging into extracellular vesicles (endo-EVs: secreted after the endocytosis of LNP-mRNA). Endosomal escape of LNP-mRNA is dependent on the molar ratio between ionizable lipids and mRNA nucleotides. Our results show that fractions of ionizable lipids and mRNA (1:1 molar ratio of hEPO mRNA nucleotides:ionizable lipids) of endocytosed LNPs were detected in endo-EVs. Importantly, these EVs can protect the exogenous mRNA during in vivo delivery to produce human protein in mice, detected in plasma and organs. Compared to LNPs, endo-EVs cause lower expression of inflammatory cytokines. Lipid nanoparticles (LNPs) are potential platforms for RNA-based therapeutics, but the fate of LNP-RNAs upon internalization into the cell is unclear. Here, the authors show that LNP-mRNAs and ionizable lipids escape the endosomes and are re-released via extracellular vesicles which could deliver the functional mRNA to other cells.

Tofacitinib, a janus kinase inhibitor, is a novel immunosuppressive drug for treatment of rheumatoid arthritis (RA). Septic arthritis (SA) and sepsis caused by Staphylococcus aureus ( S. aureus ), for which RA patients are at risk, are infections with high mortality. The aim of this study was to investigate the effect of tofacitinib on S. aureus infections using mouse models. In vitro tofacitinib treated mouse splenocytes were stimulated with S. aureus derived stimuli. Mice pre-treated with tofacitinib were inoculated intravenously with either arthritogenic- or septic doses of S. aureus . Arthritis severity and mortality were compared between groups. Additionally, pre-treated mice were challenged with staphylococcal toxin TSST-1 to induce shock. Tofacitinib inhibited splenocyte proliferation and IFN-γ production in response to TSST-1 and dead S. aureus . In SA, tofacitinib treatment aggravated arthritis with more severe bone erosions. However, in sepsis, treated mice displayed significantly prolonged survival compared to controls. Similarly, in staphylococcal enterotoxin-induced shock tofacitinib pre-treatment, but not late treatment dramatically reduced mortality, which was accompanied by decreased levels of TNF-α and IFN-γ. Our findings show that tofacitinib treatment increase susceptibility of SA in mice, but has a positive effect on survival in S. aureus -induced sepsis and a strong protective effect in toxin-induced shock.

Staphylococcus aureus biofilms, a leading cause of persistent infections, are highly resistant to immune defenses and antimicrobial therapies. In the present study, we investigated the contribution of fibrin and staphylokinase (Sak) to biofilm formation. In both clinical S. aureus isolates and laboratory strains, high Sak-producing strains formed less biofilm than strains that lacked Sak, suggesting that Sak prevents biofilm formation. In addition, Sak induced detachment of mature biofilms. This effect depended on plasminogen activation by Sak. Host-derived fibrin, the main substrate cleaved by Sak-activated plasminogen, was a major component of biofilm matrix, and dissolution of this fibrin scaffold greatly increased susceptibility of biofilms to antibiotics and neutrophil phagocytosis. Sak also attenuated biofilm-associated catheter infections in mouse models. In conclusion, our results reveal a novel role for Sak-induced plasminogen activation that prevents S. aureus biofilm formation and induces detachment of existing biofilms through proteolytic cleavage of biofilm matrix components.

Rapid bone destruction often leads to permanent joint dysfunction in patients with septic arthritis, which is mainly caused by Staphylococcus aureus (S. aureus). Staphylococcal cell wall components are known to induce joint inflammation and bone destruction. Here, we show that a single intra-articular injection of S. aureus lipoproteins (Lpps) into mouse knee joints induced chronic destructive macroscopic arthritis through TLR2. Arthritis was characterized by rapid infiltration of neutrophils and monocytes. The arthritogenic effect was mediated mainly by macrophages/monocytes and partially via TNF-α but not by neutrophils. Surprisingly, a S. aureus mutant lacking Lpp diacylglyceryl transferase (lgt) caused more severe joint inflammation, which coincided with higher bacterial loads of the lgt mutant in local joints than those of its parental strain. Coinjection of pathogenic S. aureus LS-1 with staphylococcal Lpps into mouse knee joints caused improved bacterial elimination and diminished bone erosion. The protective effect of the Lpps was mediated by their lipid moiety and was fully dependent on TLR2 and neutrophils. The blocking of CXCR2 on neutrophils resulted in total abrogation of the protective effect of the Lpps. Our data demonstrate that S. aureus Lpps elicit innate immune responses, resulting in a double-edged effect. On the one hand, staphylococcal Lpps boost septic arthritis. On the other hand, Lpps act as adjuvants and activate innate immunity, which could be useful for combating infections with multiple drug-resistant strains.

Permanent joint dysfunction is a devastating complication in patients with septic arthritis. Staphylococcus aureus ( S. aureus ) lipoproteins (Lpp), the predominant ligands for TLR2, are known to be arthritogenic and induce bone destruction when introduced directly into the joint. Here, we aim to investigate the importance of S. aureus Lpp and TLR2 in a hematogenous septic arthritis model, which is the most common route of infection in humans. C57BL/6 wild-type and TLR2 deficient mice were intravenously inoculated with S. aureus Newman parental strain or its lipoprotein-deficient Δlgt mutant strain. The clinical course of septic arthritis, radiological changes, and serum levels of cytokines and chemokines, were assessed. Newman strain induced more severe and frequent clinical septic polyarthritis compared to its Δlgt mutant in TLR2 deficient mice, but not in wild-type controls. Bone destruction, however, did not differ between groups. Lpp expression was associated with higher mortality, weight loss as well as impaired bacterial clearance in mouse kidneys independent of TLR2. Furthermore, Lpp expression induced increased systemic pro-inflammatory cytokine and neutrophil chemokine release. Staphylococcal Lpp are potent virulence factors in S. aureus systemic infection independent of host TLR2 signalling. However, they have a limited impact on bone erosion in hematogenous staphylococcal septic arthritis.

Immunosuppressive regulatory cells (IRCs) play important roles in negatively regulating immune response, and are mainly divided into myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Large numbers of preclinical and clinical studies have shown that inhibition or reduction of IRCs could effectively elevate antitumor immune responses. However, several studies also reported that excessive inhibition of IRCs function is one of the main reasons causing the side effects of cancer immunotherapy. Therefore, the reasonable regulation of IRCs is crucial for improving the safety and efficiency of cancer immunotherapy. In this review, we summarised the recent research advances in the cancer immunotherapy by regulating the proportion of IRCs, and discussed the roles of IRCs in regulating tumour immune evasion and drug resistance to immunotherapies. Furthermore, we also discussed how to balance the potential opportunities and challenges of using IRCs to improve the safety of cancer immunotherapies.

Septic arthritis remains one of the most dangerous joint diseases with a rapidly progressive disease character. Despite advances in the use of antibiotics, permanent reductions in joint function due to joint deformation and deleterious contractures occur in up to 50% of patients with septic arthritis. So far, it is still largely unknown how S. aureus initiates and establishes joint infection. Here, we demonstrate that von Willebrand factor-binding protein expressed by S. aureus facilitates the initiation of septic arthritis. Such effect might be mediated through its interaction with a host factor (von Willebrand factor). Our finding contributes significantly to the full understanding of septic arthritis etiology and will pave the way for new therapeutic modalities for this devastating disease. Septic arthritis, one of the most dangerous joint diseases, is predominantly caused by Staphylococcus aureus . In contrast, coagulase-negative staphylococci are rarely found in septic arthritis. We hypothesize that coagulases released by S. aureus , including coagulase (Coa) and von Willebrand factor-binding protein (vWbp), play potent roles in the induction of septic arthritis. Four isogenic S. aureus strains differing in expression of coagulases (wild-type [WT] Newman, Δ coa , Δ vwb , and Δ coa Δ vwb ) were used to induce septic arthritis in both wild-type and von Willebrand factor (vWF)-deficient mice. Septic arthritis severity was greatly reduced when wild-type mice were infected with the Δ coa Δ vwb and Δ vwb variants compared to WT or Δ coa strains, suggesting that vWbp rather than Coa is a major virulence factor in S. aureus septic arthritis. vWF-deficient mice were more susceptible to bone damage in septic arthritis, especially when the Δ vwb strain was used. Importantly, no difference in arthritis severity between the Δ vwb and WT strains was observed in vWF-deficient mice. Collectively, we conclude that vWbp production by S. aureus enhances staphylococcal septic arthritis. IMPORTANCE Septic arthritis remains one of the most dangerous joint diseases with a rapidly progressive disease character. Despite advances in the use of antibiotics, permanent reductions in joint function due to joint deformation and deleterious contractures occur in up to 50% of patients with septic arthritis. So far, it is still largely unknown how S. aureus initiates and establishes joint infection. Here, we demonstrate that von Willebrand factor-binding protein expressed by S. aureus facilitates the initiation of septic arthritis. Such effect might be mediated through its interaction with a host factor (von Willebrand factor). Our finding contributes significantly to the full understanding of septic arthritis etiology and will pave the way for new therapeutic modalities for this devastating disease.

Interleukin-1 receptor antagonist (IL-1Ra) is the primary therapy against autoinflammatory syndromes with robust efficacy in reducing systemic inflammation and associated organ injury. However, patients receiving IL-1Ra might be at increased risk of acquiring serious infections. To study whether IL-1Ra treatment deteriorates Staphylococcus aureus (S. aureus) septic arthritis and sepsis in mice. NMRI mice were treated with anakinra (IL-1Ra) daily for 7 days before intravenous inoculation with S. aureus strain Newman in both arthritogenic and lethal doses. The clinical course of septic arthritis, histopathological and radiological changes of the joints, as well as the mortality were compared between IL-1Ra treated and control groups. IL-1Ra treated mice developed more frequent and severe clinical septic arthritis. Also, the frequency of polyarthritis was significantly higher in the mice receiving IL-1Ra therapy. In line with the data from clinical arthritis, both histological and radiological signs of septic arthritis were more pronounced in IL-1Ra treated group compared to controls. Importantly, the mortality of IL-1Ra treated mice was significantly higher than PBS treated controls. IL-1Ra treatment significantly aggravated S. aureus induced septic arthritis and increased the mortality in these mice.

Despite being a major bacterial factor in alerting the human immune system, the role of Staphylococcus aureus (S. aureus) lipoproteins (Lpp) in skin infections remains largely unknown. Here, we demonstrated that subcutaneous injection of S. aureus Lpp led to infiltration of neutrophils and monocytes/macrophages and induced skin lesions in mice. Lipid-moiety of S. aureus Lpp and host TLR2 was responsible for such effect. Lpp-deficient S. aureus strains exhibited smaller lesion size and reduced bacterial loads than their parental strains; the altered phenotype in bacterial loads was TLR2-independent. Lpp expression in skin infections contributed to imbalanced local hemostasis toward hypercoagulable state. Depletion of leukocytes or fibrinogen abrogated the effects induced by Lpp in terms of skin lesions and bacterial burden. Our data suggest that S. aureus Lpp induce skin inflammation and promote abscess formation that protects bacteria from innate immune killing. This suggests an intriguing bacterial immune evasion mechanism.Mohammad et al. show that subcutaneous injection of Staphylococcus aureus lipoproteins (Lpp) leads to the infiltration of neutrophils and monocytes/macrophages, inducing skin lesions in mice. They find that S. aureus Lpp promotes abscess formation, which protects bacteria from innate immune killing, suggesting an intriguing bacterial immune evasion mechanism.

Lack of receptor for advanced glycation end products (RAGE) ameliorates several infections including Staphylococcus aureus pneumonia. We sought to investigate the role of RAGE in staphylococcal skin infection in mice. Wild-type (WT) and RAGE deficient (RAGE-/-) mice were subcutaneously inoculated with S. aureus SH1000 strain in abscess-forming dose or necrotic dose. Clinical signs of dermatitis, along with histopathological changes, were compared between the groups. The skin lesion size was smaller in RAGE-/- mice. Infected RAGE-/- mice expressed lower proinflammatory cytokines in local skins compared to control mice. Low dose of bacteria caused more abscess formation in RAGE-/- mice compared to skin necrosis that was more often observed in WT mice. As a result of more abscess formation, the wound healing was prolonged in RAGE-/- mice. Importantly, RAGE-/- mice had lower bacterial loads in the skin than controls, which is correlated with higher local levels of myeloperoxidase before skin infection. In vitro, enhanced phagocytic capacity of neutrophils and macrophages obtained from RAGE-/- mice compared to control mice was observed. RAGE deficiency up-regulates phagocytic capacity of phagocytes, resulting in lower bacterial burden in local skin and milder skin lesions in mice with staphylococcal skin infection.