SLAMF1-derived peptide exhibits cardio protection after permanent left anterior descending artery ligation in mice.

Acute myocardial infarction (MI) results in tissue damage to affected areas of the myocardium. The initial inflammatory response is the most damaging for residual cardiac function, while at later stages inflammation is a prerequisite for proper healing and scar formation. Balancing the extent and duration of inflammation during various stages after MI is thus pivotal for preserving cardiac function. Recently, a signaling lymphocytic activation molecule 1 (SLAMF1)-derived peptide (P7) was shown to reduce the secretion of inflammatory cytokines and protected against acute lipopolysaccharide-induced death in mice. In the present study, we experimentally induced MI by permanent ligation of the left anterior descending artery (LAD) in mice and explored the beneficial effect of immediately administering P7, with the aim of dampening the initial inflammatory phase without compromising the healing and remodeling phase. Blood samples taken 9 h post-LAD surgery and P7 administration dampened the secretion of inflammatory cytokines, but this dampening effect of P7 was diminished after 3 days. Echocardiography revealed less deterioration of cardiac contraction in mice receiving P7. In line with this, less myocardial damage was observed histologically in P7-treated mice. In conclusion, the administration of a SLAMF1-derived peptide (P7) immediately after induction of MI reduces the initial myocardial inflammation, reduces infarct expansion, and leads to less deterioration of cardiac contraction.

SARS-CoV-2 activates the TLR4/MyD88 pathway in human macrophages: A possible correlation with strong pro-inflammatory responses in severe COVID-19.

Background: Toll-like receptors (TLRs) play a pivotal role in the immunologic response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Exaggerated inflammatory response of innate immune cells, however, may drive morbidity and death in Coronavirus disease 19 (COVID-19). Objective: We investigated the engagement of SARS-CoV-2 with TLR4 in order to better understand how to tackle hyperinflammation in COVID-19. Methods: We combined RNA-sequencing data of human lung tissue and of bronchoalveolar lavage fluid cells derived from COVID-19 patients with functional studies in human macrophages using SARS-CoV-2 spike proteins and viable SARS-CoV-2. Pharmacological inhibitors as well as gene editing with CRISPR/Cas9 were used to delineate the signalling pathways involved. Results: We found TLR4 to be the most abundantly upregulated TLR in human lung tissue irrespective of the underlying pathology. Accordingly, bronchoalveolar lavage fluid cells from patients with severe COVID-19 showed an NF-κB-pathway dominated immune response, whereas they were mostly defined by type I interferon signalling in moderate COVID-19. Mechanistically, we found the Spike ectodomain, but not receptor binding domain monomer to induce TLR4-dependent inflammation in human macrophages. By using pharmacological inhibitors as well as CRISPR/Cas9 deleted macrophages, we identify SARS-CoV-2 to engage canonical TLR4-MyD88 signalling. Importantly, we demonstrate that TLR4 blockage prevents exaggerated inflammatory responses in human macrophages infected with different SARS-CoV-2 variants, including immune escape variants B.1.1.7.-E484K and B.1.1.529 (omicron). Conclusion: Our study critically extends the current knowledge on TLR-mediated hyperinflammatory responses to SARS-CoV-2 in human macrophages, paving the way for novel approaches to tackle severe COVID-19. Take-home message: Our study combining human lung transcriptomics with functional studies in human macrophages clearly supports the design and development of TLR4 - directed therapeutics to mitigate hyperinflammation in severe COVID-19.

Peptide derived from SLAMF1 prevents TLR4-mediated inflammation in vitro and in vivo.

Inflammation plays a crucial role in the development and progression of many diseases, and is often caused by dysregulation of signalling from pattern recognition receptors, such as TLRs. Inhibition of key protein-protein interactions is an attractive target for treating inflammation. Recently, we demonstrated that the signalling lymphocyte activation molecule family 1 (SLAMF1) positively regulates signalling downstream of TLR4 and identified the interaction interface between SLAMF1 and the TLR4 adaptor protein TRIF-related adapter molecule (TRAM). Based on these findings, we developed a SLAMF1-derived peptide, P7, which is linked to a cell-penetrating peptide for intracellular delivery. We found that P7 peptide inhibits the expression and secretion of IFNß and pro-inflammatory cytokines (TNF, IL-1ß, IL-6) induced by TLR4, and prevents death in mice subjected to LPS shock. The mechanism of action of P7 peptide is based on interference with several intracellular protein-protein interactions, including TRAM-SLAMF1, TRAM-Rab11FIP2, and TIRAP-MyD88 interactions. Overall, P7 peptide has a unique mode of action and demonstrates high efficacy in inhibiting TLR4-mediated signalling in vitro and in vivo.

TIRAP/Mal Positively Regulates TLR8-Mediated Signaling via IRF5 in Human Cells.

Toll-like receptor 8 (TLR8) recognizes single-stranded RNA of viral and bacterial origin as well as mediates the secretion of pro-inflammatory cytokines and type I interferons by human monocytes and macrophages. TLR8, as other endosomal TLRs, utilizes the MyD88 adaptor protein for initiation of signaling from endosomes. Here, we addressed the potential role of the Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) in the regulation of TLR8 signaling in human primary monocyte-derived macrophages (MDMs). To accomplish this, we performed TIRAP gene silencing, followed by the stimulation of cells with synthetic ligands or live bacteria. Cytokine-gene expression and secretion were analyzed by quantitative PCR or Bioplex assays, respectively, while nuclear translocation of transcription factors was addressed by immunofluorescence and imaging, as well as by cell fractionation and immunoblotting. Immunoprecipitation and Akt inhibitors were also used to dissect the signaling mechanisms. Overall, we show that TIRAP is recruited to the TLR8 Myddosome signaling complex, where TIRAP contributes to Akt-kinase activation and the nuclear translocation of interferon regulatory factor 5 (IRF5). Recruitment of TIRAP to the TLR8 signaling complex promotes the expression and secretion of the IRF5-dependent cytokines IFNß and IL-12p70 as well as, to a lesser degree, TNF. These findings reveal a new and unconventional role of TIRAP in innate immune defense.

SLAMF1 is required for TLR4-mediated TRAM-TRIF-dependent signaling in human macrophages.

Signaling lymphocytic activation molecule family 1 (SLAMF1) is an Ig-like receptor and a costimulatory molecule that initiates signal transduction networks in a variety of immune cells. In this study, we report that SLAMF1 is required for Toll-like receptor 4 (TLR4)-mediated induction of interferon ß (IFNß) and for killing of Gram-negative bacteria by human macrophages. We found that SLAMF1 controls trafficking of the Toll receptor-associated molecule (TRAM) from the endocytic recycling compartment (ERC) to Escherichia coli phagosomes. In resting macrophages, SLAMF1 is localized to ERC, but upon addition of E. coli, it is trafficked together with TRAM from ERC to E. coli phagosomes in a Rab11-dependent manner. We found that endogenous SLAMF1 protein interacted with TRAM and defined key interaction domains as amino acids 68 to 95 of TRAM as well as 15 C-terminal amino acids of SLAMF1. Interestingly, the SLAMF1-TRAM interaction was observed for human but not mouse proteins. Overall, our observations suggest that SLAMF1 is a new target for modulation of TLR4-TRAM-TRIF inflammatory signaling in human cells.

The role of CD150-SH2D1A association in CD150 signaling in Hodgkin's lymphoma cell lines.

AIM: To find out what signal transduction pathways are linked to CD150 in Hodgkin's lymphoma (HL) cell lines, how are they regulated, and to examine the expression of different CD150 splice forms and SH2 domain containing protein D1A (SH2D1A) adaptor protein on mRNA level in HD primary tumors and cell lines. METHODS: The expression of CD150 splicing forms and SH2D1A adaptor protein in HD primary lymphoma tissue and cell lines were analyzed by RT-PCR method. To examine CD150-SH2D1A localization in HD cell lines we performed double immunofluorescent staining of these two proteins. Total amount of SH2D1A, Syk, Lyn, SHP-2, SHIP proteins, and activated/phosphorylated ERK1/2 and Akt proteins were detected by Western blot analysis with specific antibodies. RESULTS: We showed the expression of soluble (sCD150) and full length transmembrane (mCD150) splice forms and SH2D1A adaptor protein on mRNA level in 9 cases of classical HD and three HD lines of B cell origin - L428, KM-H2 and L1236. In spite of CD150 and SH2D1A co-expression in studied HD cell lines, CD150 co-precipitated and co-localized with SH2D1A only in L1236 cells. CD150 ligation induced extracellular signal-regulated kinases (ERK) dephosphorylation in L1236 cell line, but had no effect on ERK pathway in KM-H2 and L428 cells. CD150 crosslinking induced Akt activation also only in L1236 cells. CONCLUSIONS: HD cells express sCD150 and mCD150 splice forms and SH2D1A. Association of CD150 and SH2D1A depends at least on their localization pattern. CD150 is linked to ERK and Akt pathways in HD cell lines. Our data suggest that CD150-SH2D1A association play decisive role in Akt signaling upon CD150 ligation in HD cell lines. CD150-mediated Akt activation in HD cell lines, similarly to DT40 model system, is SHIP-independent.

The adaptor protein SH2D1A regulates signaling through CD150 (SLAM) in B cells.

The CD150 receptor is expressed on activated T and B lymphocytes, dendritic cells, and monocytes. A TxYxxV/I motif in the CD150 cytoplasmic tail can bind different SH2-containing molecules, including tyrosine and inositol phosphatases, Src family kinases, and adaptor molecules. To analyze CD150-initiated signal transduction pathways, we used DT40 B-cell sublines deficient in these molecules. CD150 ligation on DT40 transfectants induced the extracellular signal-regulated kinase (ERK) pathway, which required SH2-containing inositol phosphatase (SHIP) but not SH2 domain protein 1A (SH2D1A). CD150-mediated Akt phosphorylation required Syk and SH2D1A, was negatively regulated by Lyn and Btk, but was SHIP independent. Lyn directly phosphorylated Y327 in CD150, but the Akt pathway did not depend on CD150 tyrosine phosphorylation and CD150-SHP-2 association. Analysis of CD150 and SH2D1A expression in non-Hodgkin and Hodgkin lymphomas revealed stages of B-cell differentiation where these molecules are expressed alone or coexpressed. Signaling studies in Hodgkin disease cell lines showed that CD150 is linked to the ERK and Akt pathways in neoplastic B cells. Our data support the hypothesis that CD150 and SH2D1A are coexpressed during a narrow window of B-cell maturation and SH2D1A may be involved in regulation of B-cell differentiation via switching of CD150-mediated signaling pathways.