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Liposomes are frequently used in cancer therapy to encapsulate and apply anticancer drugs. Here, we show that a systemic treatment of mice bearing skin tumors with empty phosphatidylcholine liposomes (PCL) resulted in inhibition of tumor growth, which was similar to that observed with the synthetic bacterial lipoprotein and TLR1/2 agonist Pam(3)CSK(4) (BLP). Both compounds led to a substantial decrease of macrophages in spleen and in the tumor-bearing skin. Furthermore, both treatments induced the expression of typical macrophage markers in the tumor-bearing tissue. As expected, BLP induced the expression of the M1 marker genes Cxcl10 and iNOS, whereas PCL, besides inducing iNOS, also increased the M2 marker genes Arg1 and Trem2. In vitro experiments demonstrated that neither PCL nor BLP influenced proliferation or survival of tumor cells, whereas both compounds inhibited proliferation and survival and increased the migratory capacity of bone marrow-derived macrophages (BMDM). However, in contrast to BLP, PCL did not activate cytokine secretion and induced a different BMDM phenotype. Together, the data suggest that similar to BLP, PCL induce an antitumor response by influencing the tumor microenvironment, in particular by functional alterations of macrophages, however, in a distinct manner from those induced by BLP.

Successful cancer immunotherapy entails activation of innate immune receptors to promote dendritic cell (DC) maturation, antigen presentation, up-regulation of costimulatory molecules, and cytokine secretion, leading to activation of tumor antigen-specific cytotoxic T lymphocytes (CTLs). Here we screened a synthetic library of 100,000 compounds for innate immune activators using TNF production by THP-1 cells as a readout. We identified and optimized a potent human and mouse Toll-like receptor (TLR)1/TLR2 agonist, Diprovocim, which exhibited an EC50 of 110 pM in human THP-1 cells and 1.3 nM in primary mouse peritoneal macrophages. In mice, Diprovocim-adjuvanted ovalbumin immunization promoted antigen-specific humoral and CTL responses and synergized with anti–PD-L1 treatment to inhibit tumor growth, generating long-term antitumor memory, curing or prolonging survival of mice engrafted with the murine melanoma B16-OVA. Diprovocim induced greater frequencies of tumor-infiltrating leukocytes than alum, of which CD8 T cells were necessary for the antitumor effect of immunization plus anti–PD-L1 treatment.

Fibroblasts play a pivotal role in key processes within the heart, particularly in cardiac remodeling that follows both ischemic and non-ischemic injury. During remodeling, fibroblasts drive fibrosis and inflammation by reorganizing the extracellular matrix and modulating the immune response, including toll-like receptor (TLR) activation, to promote tissue stabilization. Building on findings from our prior research on heart tissue from patients with advanced coronary artery disease and aortic valve disease, this study sought to explore specific effects of TLR1, TLR3, and TLR7 activation on NF-κB signaling, proinflammatory cytokine production, and γ-protocadherin expression in cardiac fibroblasts. Human cardiac fibroblasts were exposed to agonists for TLR1, TLR3, or TLR7 for 24 h, followed by an analysis of NF-κB signaling, cytokine production, and γ-protocadherin expression. The activation of these TLRs triggered distinct responses in the NF-κB signaling pathway, with TLR3 showing a stronger activation profile compared to TLR1 and TLR7, particularly in downregulating γ-protocadherin expression. These findings highlight a potential role for TLR3 in amplifying inflammatory responses and reducing γ-protocadherin levels in cardiac fibroblasts, correlating with the enhanced inflammation and lower γ-protocadherin expression observed in diseased myocardium from patients with coronary artery disease and aortic valve disease. Consequently, TLR3 represents a potential therapeutic target for modulating immune responses in cardiovascular diseases.

Toll-like receptor (TLRs) activation in multiple myeloma (MM) cells induces heterogeneous functional responses including cell growth and proliferation, survival or apoptosis. These effects have been suggested to be partly due to increase in secretion of cytokines such as IL-6 or IFNα among others from MM cells following TLR activation. However, whether triggering of these receptors also modulates production of immunoglobulin free light chains (FLCs), which largely contribute to MM pathology, has not been investigated in MM cells before. This study explored the effect of TLR1/2 ligand (Pam3CSK4) alone or combined with bortezomib (BTZ) on production of FLCs in human myeloma cell lines, L363, OPM-2, U266 and NCI-H929. It also investigated the above effect when MM cells were exposed to bone marrow stromal cells (BMSCs) or fibronectin (FN). Adhesion to BMSCs or FN increased secretion of FLC in MM cells. Pam3CSK4 decreased FLC production, and this effect was enhanced in combination with BTZ but attenuated when MM cells adhered to BMSCs or FN. The findings of this study imply that activation of TLR1/2 downregulates FLC production in MM cells even in the context of bone marrow microenvironment components and suggest that targeting some TLRs such as TLR1/2 might have therapeutic potential.

Toll-like receptors (TLRs) are activated by endogenous molecules released from damaged cells and contribute to neuroinflammation following traumatic brain injury (TBI) and epilepsy. TLR1/2 agonist tri-palmitoyl-S-glyceryl-cysteine (Pam3cys) is a vaccine adjuvant with confirmed safety in humans. We assessed impact of TLR1/2 postconditioning by Pam3cys on epileptogenesis and neuroinflammation in male rats, 6, 24, and 48 h after mild-to-moderate TBI. Pam3cys was injected into cerebral ventricles 30 min after controlled cortical impact (CCI) injury. After 24 h, rats underwent chemical kindling by once every other day injections of pentylenetetrazole (PTZ) 35 mg/kg until development of generalized seizures. Number of intact neurons, brain expression of proinflammatory cytokine TNF-α, anti-inflammatory cytokine IL-10, and marker of anti-inflammatory microglia arginase1 (Arg1) were determined by immunoblotting. Astrocytes and macrophage/microglia activation/polarization at the contused area was assessed by double immunostaining with Iba1/Arg1, Iba1/iNOS and GFAP/iNOS, specific antibodies. The CCI-injured rats became kindled by less number of PTZ injections than sham-operated rats (9 versus 14 injections, p  < 0.0001). Pam3cys treatment returned the accelerated rate of epileptogenesis in TBI state to the sham level. Pam3cys decreased neural death 48 h after TBI. It decreased TNF-α (6 h post-TBI, p  < 0.01), and up-regulated IL-10 ( p  < 0.01) and Arg1 ( p  < 0.05) 48 h after TBI. The iNOS-positive cells decreased ( p  < 0.001) whereas Iba1/Arg1-positive cells enhanced ( p  < 0.01) after Pam3cys treatment. Pam3cys inhibits TBI-accelerated acquisition of seizures. Pam3cys reprograms microglia and up-regulates anti-inflammatory cytokines during the first few days after TBI. This capacity along with the clinical safety, makes Pam3cys a potential candidate for development of effective medications against posttraumatic epilepsy.

Background Toll-like receptors (TLRs) are crucial for recognition of pathogen-associated molecular patterns by cells of the innate immune system. TLRs are present and functional in CD4 + T cells. Memory CD4 + T cells, predominantly central memory cells (T CM ), constitute the main reservoir of latent HIV-1. However, how TLR ligands affect the quiescence of latent HIV within central memory CD4 + T cells has not been studied. Results We evaluated the ability of a broad panel of TLR agonists to reactivate latent HIV-1. The TLR-1/2 agonist Pam3CSK4 leads to viral reactivation of quiescent HIV in a model of latency based on cultured T CM and in resting CD4 + T cells isolated from aviremic patients. In addition, we investigated the signaling pathway associated with Pam3CSK4 involved in HIV-1 reactivation. We show that the transcription factors NFκB, NFAT and AP-1 cooperate to induce viral reactivation downstream of TLR-1/2 stimulation. Furthermore, increasing levels of cyclin T1 is not required for TLR-mediated viral reactivation, but induction of viral expression requires activated pTEFb. Finally, Pam3CSK4 reactivates latent HIV-1 in the absence of T cell activation or proliferation, in contrast to antigen stimulation. Conclusions Our findings suggest that the signaling through TLR-1/2 pathway via Pam3CSK4 or other reagents should be explored as an anti-latency strategy either alone or in combination with other anti-latency drugs.

Toll-like receptor (TLR)-mediated innate immune responses are important in early host defense. Using a candidate gene approach, we previously identified genetic variation within TLR1 that is associated with hyper-responsiveness to a TLR1/2 agonist in vitro and with death and organ dysfunction in patients with sepsis. Here we report a genome-wide association study (GWAS) designed to identify genetic loci controlling whole blood cytokine responses to the TLR1/2 lipopeptide agonist, Pam 3 CSK 4 (N-palmitoyl-S-dipalmitoylglyceryl Cys-Ser-(Lys) 4 ) ex vivo . We identified a very strong association ( P <1 × 10 −27 ) between genetic variation within the TLR10/1/6 locus on chromosome 4, and Pam 3 CSK 4 -induced cytokine responses. This was the predominant association explaining over 35% of the population variance for this phenotype. Notably, strong associations were observed within TLR10 , suggesting that genetic variation in TLR10 may influence bacterial lipoprotein-induced responses. These findings establish the TLR10/1/6 locus as the dominant common genetic factor controlling interindividual variability in Pam 3 CSK 4 -induced whole blood responses in the healthy population.

Toll-like receptors (TLRs) constitute a family of nonpolymorphic receptors that are devoted to pathogen recognition. In this work, we have explored the impact of TLR ligands (TLR-L) on human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). We show that HSCs and HPCs have a comparable pattern of expression of TLR transcripts characterized by the predominance of TLR1, -2, -3, -4 and -6. In long-term cultures of HSCs, HPCs and stromal cells, most TLR-L profoundly inhibited B-cell development while preserving or enhancing the production of myeloid cells. In short-term cultures, the TLR1/2 ligand PAM(3)CSK(4) induced a large proportion of HPCs to express markers of the myelomonocytic lineage. PAM(3)CSK(4) induced only marginal expression of myeloid lineage markers on HSCs but promoted their myeloid commitment as revealed by their acquisition of the phenotype of multi- and bipotential myeloid progenitors and by upregulation of the transcription factors PU.1, C/EBPalpha and GATA-1. Our results suggest that TLR agonists can bias the lineage commitment of human HSCs and shift the differentiation of lineage-committed progenitors to favor myelopoiesis at the expense of lymphoid B-cell development.

Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are major respiratory pathogens and can concurrently colonize the airways of patients with chronic obstructive diseases, such as cystic fibrosis (CF). Airway epithelial cell signalling is critical to the activation of innate immune responses. In the setting of polymicrobial colonization or infection of the respiratory tract, how epithelial cells integrate different bacterial stimuli remains unknown. Our study examined the inflammatory responses to PA and SA co-stimulations. Immortalised airway epithelial cells (Beas-2B) exposed to bacteria-free filtrates from PA (PAF) induced a robust production of the neutrophil chemoattractant IL-8 while bacteria-free filtrates from SA (SAF) had a minimal effect. Surprisingly, co-stimulation with PAF+SAF demonstrated that SAF strongly inhibited the PAF-driven IL-8 production, showing that SAF has potent anti-inflammatory effects. Similarly SAF decreased IL-8 production induced by the TLR1/TLR2 ligand Pam3CysSK4 but not the TLR4 ligand LPS nor TLR5 ligand flagellin in Beas-2B cells. Moreover, SAF greatly dampened TLR1/TLR2-mediated activation of the NF-κB pathway, but not the p38 MAPK pathway. We observed this SAF-dependent anti-inflammatory activity in several SA clinical strains, as well as in the CF epithelial cell line CFBE41o-. These findings show a novel direct anti-inflammatory effect of SA on airway epithelial cells, highlighting its potential to modulate inflammatory responses in the setting of polymicrobial infections.

BACKGROUND: Effects of Giardia duodenalis on dendritic cell (DC) functions may contribute to the pathogenesis of chronic giardiasis. G. duodenalis lysate has been shown to inhibit the activation of murine DCs through the ligands of various Toll-like receptors (TLRs), including TLR2 and TLR4. Our study aimed at translating these findings to human DCs. FINDINGS: As described previously for murine DCs, also human DCs were only weakly activated by the parasite itself. LPS-stimulated DCs incubated in the presence of G. duodenalis lysate produced less IL-12/23p40 (p = 0.002), IL-12p70 (p = 0.011), and IL-23 (p = 0.004), but more IL-10 (p = 0.006) than cells incubated in the absence of the parasite. Concomitantly, the expression of CD25, CD83, CD86, and HLA-DR was reduced on G. duodenalis-incubated DCs as compared to control cells. In contrast, human DCs stimulated through TLR2 in combination with TLR1 or TLR6 and G. duodenalis lysate secreted significantly more IL-12/23p40 (p = 0.006), IL-23 (p = 0.002), and IL-10 (p = 0.014) than cells stimulated through TLR2 ligands alone. Ligands for TLR2/TLR1 or TLR2/TLR6 also induced enhanced extracellular expression of CD25, CD83, and CD86 (p < 0.05). CONCLUSIONS: In contrast to murine DCs, human DCs incubated in the presence of G. duodenalis and stimulated through TLR2 show increased activation as compared to cells incubated in the absence of the parasite. Thus, TLR2 ligands, e.g., delivered by probiotic lactobacilli, might be beneficial in human giardiasis through an adjuvant effect on the induction of cellular immune responses against G. duodenalis.