BACKGROUND: Triple-negative breast cancer (TNBC) is prone to relapse due to the lack of effective therapeutic targets. Macrophages are the most abundant immune cells in the tumor microenvironment (TME) of breast cancer. Targeting the cross-talk between macrophages and cancer cells provides a more efficient strategy for anti-tumor therapy. Toll-like receptors (TLRs) are important players involved in macrophage activation, and TLR agonists are known to play roles in cancer therapy. However, the combination strategy of TLR agonists with chemotherapy drugs is still not well characterized.
METHODS: RT-PCR and Western blot were used to detect the expression of TLRs. The communication between breast cancer cells and macrophages were determined by co-culture in vitro. Tumor cells proliferation and migration were investigated by MTT assay and scratch wound assay. The effects of drug combinations and toxic side effects were assessed by immunohistochemistry and Hematoxylin & Eosin staining.
RESULTS: Expression of TLR3 and TLR4 were lower in breast tumor tissues compared with adjacent normal tissues. Patients with higher TLR3 or TLR4 expression levels had a better prognosis than those with lower expression levels. TLR3/4 expression was significantly inhibited when breast cancer cells MDA-MB-231 and E0771 were conditioned-cultured with macrophages in vitro and was also inhibited by pirarubicin (THP). However, the combination of TLR agonists and THP could reverse this response and inhibit the proliferation and migration of breast cancer cells. Additionally, this combination significantly reduced the tumor volume and weight in the murine model, increased the expression of TLR3/4 in mouse breast tumors.
CONCLUSIONS: Our results provide new ideas for the combination strategy of THP with TLR agonists which improves prognosis of breast cancer.

Talaromycosis, caused by Talaromyces marneffei (T. marneffei, formerly known as Penicillium marneffei), is an opportunistic invasive mycosis endemic in tropical and subtropical areas of Asia with high mortality rate. Despite various infection models established to study the immunological interaction between T. marneffei and the host, the pathogenicity of this fungus is not yet fully understood. So far, Drosophila melanogaster, a well-established genetic model organism to study innate immunity, has not been used in related research on T. marneffei. In this study, we provide the initial characterization of a systemic infection model of T. marneffei in the D. melanogaster host. Survival curves and fungal loads were tested as well as Toll pathway activation was quantified by RT-qPCR of several antimicrobial peptide (AMP) genes including Drosomycin, Metchnikowin, and Bomanin Short 1. We discovered that whereas most wild-type flies were able to overcome the infection, MyD88 or Toll mutant flies failed to prevent fungal dissemination and proliferation and ultimately succumbed to this challenge. Unexpectedly, the induction of classical Toll pathway activation readouts, Drosomycin and Bomanin Short 1, by live or killed T. marneffei was quite limited in wild-type flies, suggesting that the fungus largely escapes detection by the systemic immune system. This unusual situation of a poor systemic activation of the Toll pathway and a strong susceptibility phenotype of MyD88/Toll might be accounted for by a requirement for this host defence in only specific tissues, a hypothesis that remains to be rigorously tested.

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Strains of the Bacillus cereus (Bc) group are sporulating bacteria commonly associated with foodborne outbreaks. Spores are dormant cells highly resistant to extreme conditions. Nevertheless, the pathological processes associated with the ingestion of either vegetative cells or spores remain poorly understood. Here, we demonstrate that while ingestion of vegetative bacteria leads to their rapid elimination from the intestine of Drosophila melanogaster, a single ingestion of spores leads to the persistence of bacteria for at least 10 days. We show that spores do not germinate in the anterior part of the intestine which bears the innate immune defenses. Consequently, spores reach the posterior intestine where they germinate and activate both the Imd and Toll immune pathways. Unexpectedly, this leads to the induction of amidases, which are negative regulators of the immune response, but not to antimicrobial peptides. Thereby, the local germination of spores in the posterior intestine dampens the immune signaling that in turn fosters the persistence of Bc bacteria. This study provides evidence for how Bc spores hijack the intestinal immune defenses allowing the localized birth of vegetative bacteria responsible for the digestive symptoms associated with foodborne illness outbreaks.

Mesenchymal stromal cells (MSCs) have evolved as an invaluable therapeutic cell type due to their broad therapeutic properties. Bone marrow-derived MSCs are currently being applied in numerous clinical trials, and the initial results have been encouraging. However, heterogeneous responsiveness amongst patients is also being experienced; therefore, the efficacy of MSCs in vivo is still debatable. Host microenvironment plays an essential role in determining the fate of MSCs in vivo. Recent studies have indicated the role of toll-like receptors (TLR) in modulating the biological properties of MSCs. TLRs are expressed by MSCs, and activation of TLR3 and TLR4 can alter the functionality of MSCs. While MSCs can suppress the effector and memory T cell function by promoting regulatory T cells, the effect of TLR activation on MSC-mediated immune cell induction is still not well understood. This study was performed to understand the TLR licensing of MSCs and its impact on MSC-mediated immunomodulation. We found that TLR3 mediated activation of MSCs (TLR3-MSCs) increased the expression of G-CSF & IL-10 while TLR4-mediated activation of MSCs led to an increase in CXCL-1, CXCL-10, and CXCL-12. To study the immunological aspect, an in vitro co-culture model was established-to imitate the brief in vivo interaction of MSCs and immune cells. We found that TLR3-MSCs led to increase in CD4 and CD8 naive T (TNAI) cells and vice versa for effector (TEFF) and memory T (TMEM) cells, while TLR4-MSCs did not show any effect. Moreover, only TLR3-MSCs led to a non-significant increase in the regulatory T cells (TREGS) and Double negative regulatory cells. No change in B cell profile was evident while TLR3-MSCs depicted an increasing trend in regulatory B cells which was not statistically significant. TLR3 MSCs also inhibited the T cell proliferation in our setup. Our data indicate that TLR3 priming may regulate the function of MSCs through immunomodulation. Understanding the role of TLRs and other microenvironmental factors causing subdued responses of MSCs in vivo would allow the uninhibited use of MSCs for many diseased conditions.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases of pigs globally. The pathogen, porcine reproductive and respiratory syndrome virus (PRRSV), is an enveloped positive-stranded RNA virus, which is considered to be the key triggers for the activation of effective innate immunity through pattern recognition receptor (PRR)-dependent signaling pathways. Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs) and Cytoplasmic DNA receptors (CDRs) are used as PRRs to identify distinct but overlapping microbial components. The innate immune system has evolved to recognize RNA or DNA molecules from microbes through pattern recognition receptors (PRRs) and to induce defense response against infections, including the production of type I interferon (IFN-I) and inflammatory cytokines. However, PRRSV is capable of continuous evolution through gene mutation and recombination to evade host immune defenses and exploit host cell mechanisms to synthesize and transport its components, thereby facilitating successful infection and replication. This review presents the research progress made in recent years in the study of these PRRs and their associated adapters during PRRSV infection.

BACKGROUND: Genetic variants may influence Toll-like receptor (TLR) signaling in the immune response to human papillomavirus (HPV) infection and lead to cervical cancer. In this study, we investigated the pattern of TLR expression in the transcriptome of HPV-positive and HPV-negative cervical cancer samples and looked for variants potentially related to TLR gene alterations in exomes from different populations.
METHODS: A cervical tissue sample from 28 women, which was obtained from the Gene Expression Omnibus database, was used to examine TLR gene expression. Subsequently, the transcripts related to the TLRs that showed significant gene expression were queried in the Genome Aggregation Database to search for variants in more than 5,728 exomes from different ethnicities.
RESULTS: Cancer and HPV were found to be associated (p<0.0001). TLR1(p = 0.001), TLR3(p = 0.004), TLR4(221060_s_at)(p = 0.001), TLR7(p = 0.001;p = 0.047), TLR8(p = 0.002) and TLR10(p = 0.008) were negatively regulated, while TLR4(1552798_at)(p<0.0001) and TLR6(p = 0.019) were positively regulated in HPV-positive patients (p<0.05). The clinical significance of the variants was statistically significant for TLR1, TLR3, TLR6 and TLR8 in association with ethnicity. Genetic variants in different TLRs have been found in various ethnic populations. Variants of the TLR gene were of the following types: TLR1(5_prime_UTR), TLR4(start_lost), TLR8(synonymous;missense) and TLR10(3_prime_UTR). The \"missense\" variant was found to have a risk of its clinical significance being pathogenic in South Asian populations (OR = 56,820[95%CI:40,206,80,299]).
CONCLUSIONS: The results of this study suggest that the variants found in the transcriptomes of different populations may lead to impairment of the functional aspect of TLRs that show significant gene expression in cervical cancer samples caused by HPV.

BACKGROUND: The problem of identifying vaccine-specific T-cell responses is still a matter of debate. Currently, there are no universal, clearly defined, agreed upon criteria for assessing the effectiveness of vaccinations and their immunogenicity for the cellular component of immunity, even for healthy people. But for patients with inborn errors of immunity (IEI), especially those with antibody deficiencies, evaluating cellular immunity holds significant importance.
OBJECTIVE: To examine the effect of one and two doses of inactivated adjuvanted subunit influenza vaccines on the expression of endosomal Toll-like receptors (TLRs) on the immune cells and the primary lymphocyte subpopulations in patients with common variable immunodeficiency (CVID).
METHODS: During 2018-2019, six CVID patients received one dose of a quadrivalent adjuvanted influenza vaccine; in 2019-2020, nine patients were vaccinated with two doses of a trivalent inactivated influenza vaccine. The proportion of key lymphocyte subpopulations and expression levels of TLRs were analyzed using flow cytometry with monoclonal antibodies.
RESULTS: No statistically significant alterations in the absolute values of the main lymphocyte subpopulations were observed in CVID patients before or after vaccination with the different immunization protocols. However, after vaccination, a higher expression of TLR3 and TLR9 in granulocytes, monocytes, and lymphocytes was found in those patients who received two vaccine doses rather than one single dose.
CONCLUSIONS: This study marks the first instance of using a simultaneous two-dose vaccination, which is associated with an elevated level of TLR expression in the immune cells. Administration of the adjuvanted vaccines in CVID patients appears promising. Further research into their impact on innate immunity and the development of more effective vaccination regimens is warranted.

In polymicrobial sepsis, the extracellular histones, mainly released from activated neutrophils, significantly contribute to cardiac dysfunction (septic cardiomyopathy), as demonstrated in our previous studies using Echo-Doppler measurements. This study aims to elucidate the roles of extracellular histones and their interactions with Toll-like receptors (TLRs) in cardiac dysfunction. Through ex vivo assessments of ECG, left ventricle (LV) function parameters, and in vivo Echo-Doppler studies in mice perfused with extracellular histones, we aim to provide comprehensive insights into the mechanisms underlying sepsis-induced cardiac dysfunction. Langendorff-perfused hearts from both wild-type and TLR2, TLR3, or TLR4 knockout (KO) mice were examined. Paced mouse hearts were perfused with histones to assess contractility and relaxation. Echo-Doppler studies evaluated cardiac dysfunction after intravenous histone injection. Histone perfusion caused defects in contractility and relaxation, with TLR2 and TLR3 KO mice being partially protected. Specifically, TLR2 KO mice exhibited the greatest reduction in Echo-Doppler abnormalities, while TLR4 KO exacerbated cardiac dysfunction. Among individual histones, H1 induced the most pronounced abnormalities in cardiac function, apoptosis of cardiomyocytes, and LDH release. Our data highlight significant interactions between histones and TLRs, providing insights into histones especially H1 as potential therapeutic targets for septic cardiomyopathy. Further studies are needed to explore specific histone-TLR interactions and their mechanisms.

Phagocytosis is a central process by which macrophage cells internalize and eliminate microbes as well as apoptotic cells. The nascent phagosome undergoes a complex maturation process involving sequential fusion with endosomal compartments. The endosomal TLRs, including TLR3, -7, -8, and -9, play a critical role in innate immunity by sensing bacterial or viral nucleic acids and are preferentially transported to the phagosomal membrane of innate immune cells upon activation. Therefore, phagosome isolation is helpful for studies on pathogenic invasion and the functions of phagosome proteins, including endosomal TLRs.