TLR Stimulation Research Articles

Preclinical study of TLR stimulation combined PD-1 antibody enhance the therapeutic effect of microwave ablation on NSCLC.

The purpose of this study was to investigate the therapeutic efficacy of the combination of microwave ablation (MWA) with immune checkpoints blockade and TLR9 stimulation in the treatment of non-small cell lung cancer (NSCLC) using the C57BL/6 tumor-bearing mice model. Tumor-bearing mice were treated with MWA, programmed cell death protein1 blockade (PD-1) plus MWA (MWA + P), TLR9 agonist CpG ODNs and MWA (MWA + C), PD-1 blockade and CpG ODNs (P + C), MWA plus PD-1 blockade and CpG ODNs (MWA + P + C), or untreated. Survival time was evaluated with the Kaplan-Meyer method comparing survival curves by log-rank test. On day 15 after MWA, ten mice from the combination therapy group received tumor rechallenge with LLC cells and the volumes of rechallenge tumor were calculated every 5days. Immune cells were identified by immunohistochemistry and flow cytometry, and the concentrations of IFN-γ、TNF-α and TGF-β were identified by enzyme-linked immunosorbent assay (ELISA). The MWA + P + C combination therapy significantly prolonged tumor-bearing mice survival and reduced tumor size compared to untreated group, MWA group, MWA + P group, M + C group, P + C group. The combination therapy also protected most surviving mice from LLC tumor rechallenge. CD8 + T-cell in tumor and spleen were remarkably induced by MWA + P + C and Treg cell further diminished by combination therapy. Both tumor necrosis factor-alpha (TNF-α) and interferon-gama (IFN-γ) concentrations in plasma were significantly elevated in the combination therapy group compared to other groups, while transforming growth factor Beta (TGF-β) was reduced. MWA combined with immune checkpoints blockade and TLR stimulation could significantly enhance antitumor efficacy with augmented specific immune responses, and the combination therapy is a promising approach to treat non-small cell lung cancer (NSCLC).

Differential TLR-ERK1/2 Activity Promotes Viral ssRNA and dsRNA Mimic-Induced Dysregulated Immunity in Macrophages.

RNA virus-induced excessive inflammation and impaired antiviral interferon (IFN-I) responses are associated with severe disease. This innate immune response, also referred to as "dysregulated immunity" is caused by viral single-stranded RNA (ssRNA)- and double-stranded-RNA (dsRNA)-mediated exuberant inflammation and viral protein-induced IFN antagonism. However, key host factors and the underlying mechanism driving viral RNA-mediated dysregulated immunity are poorly defined. Here, using viral ssRNA and dsRNA mimics, which activate toll-like receptor 7 (TLR7) and TLR3, respectively, we evaluated the role of viral RNAs in causing dysregulated immunity. We observed that murine bone marrow-derived macrophages (BMDMs), when stimulated with TLR3 and TLR7 agonists, induced differential inflammatory and antiviral cytokine response. TLR7 activation triggered a robust inflammatory cytokine/chemokine induction compared to TLR3 activation, whereas TLR3 stimulation induced significantly increased IFN/IFN stimulated gene (ISG) response relative to TLR7 activation. To define the mechanistic basis for dysregulated immunity, we examined cell-surface and endosomal TLR levels and downstream mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) activation. We identified significantly higher cell-surface and endosomal TLR7 levels compared to TLR3, which were associated with early and robust MAPK (p-ERK1/2, p-P38, and p-JNK) and NF-kB activation in TLR7-stimulated macrophages. Furthermore, blocking EKR1/2 and NF-kB activity reduced TLR3/7-induced inflammatory cytokine/chemokine levels, whereas only ERK1/2 inhibition enhanced viral RNA mimic-induced IFN/ISG responses. Collectively, our results illustrate that high cell-surface and endosomal TLR7 expression and robust ERK1/2 activation drive viral ssRNA mimic-induced excessive inflammatory and reduced IFN/ISG response and blocking ERK1/2 activity would likely mitigate viral-RNA/TLR-induced dysregulated immunity.

Abstract 4136901: Mechanosensitive channel protein Piezo1 synergizes with TLR4 to enhance osteogenic responses in human aortic valve interstitial cells

Background: Calcific aortic valve disease (CAVD), a common heart valve disease in the elderly, has a complex pathogenesis. Abnormal blood flow shear can be regarded as an important factor in the development of CAVD, and a key molecule that responds to mechanical stress is the mechanosensitive ion channel protein Piezo1; however, the role of Piezo1 in CAVD has not been clarified. Methods and results: We collected CAVD patients and their age- and sex-matched controls, and compared the differences in the protein expression levels of Piezo1 and TLR4 between the two using Western blot and other techniques, and found that the expression of Piezo1 and TLR4 proteins was elevated in the CAVD group. We gave LPS or Piezo1 agonist Yoda1 to stimulate aortic valve mesenchymal stromal cells, respectively, and the Western blot results showed that ALP and Runx2 expression were elevated after LPS and Yoda1 stimulation, and immunofluorescence and alizarin red staining showed similar results. Inhibition of TLR4 or Piezo1 attenuated the osteogenic differentiation of AVICs. Cellular immunofluorescence results showed that TLR4 could be transported from the Golgi to the cell membrane under Yoda stimulation, and Golgi-cell membrane transport of TLR4 was attenuated after Rab3b knockdown by siRNA. After first pre-stimulation with Yoda1 and then stimulation with LPS, Western blot and immunofluorescence results showed that the protein levels of ALP and Runx2 as well as NF-κB phosphorylation were to be significantly increased. Conclusion: Stimulation of Piezo1 and TLR4 can enhance the osteogenic response of AVICs. Stimulation of Piezo1 enhances the transport of TLR4 from the Golgi to the cell membrane via Rab3b, and Piezo1 synergistically enhances the osteogenic response of AVICs with TLR4, thus promoting the development of CAVD.

Loss of O6-methylguanine DNA methyltransferase (MGMT) in macrophages alters responses to TLR3 stimulation and enhances DNA double-strand breaks and mitophagy

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA damage repair enzyme. The roles of this enzyme in immune cells remain unclear. In this study, we explored the roles of MGMT in bone marrow-derived murine macrophages (BMMs) via the use of MGMT knockout (KO) mice. Loss of MGMT altered the response to TLR3 agonists (poly (I:C)), such as dampening the production of TNFα and IL-6. Increased DNA double-strand breaks (DSBs) were observed in MGMT-KO macrophages but did not result in increased cell death. MGMT localized to both nuclei and mitochondria at increasing levels during poly (I:C) stimulation. MGMT deficiency increased the production of mitochondrial reactive oxygen species (mtROS), which was correlated with increased mitophagy. The underlying mechanism involves mediation through activation of the AMPKα pathway. Taken together, our findings reveal the roles of MGMT in macrophages in regulating the response to TLR3, which links DSBs to mtROS and mitophagy via the AMPKα pathway. These roles may have consequences for the inflammatory response and chronic inflammation.

Hyperresponsiveness of Corticoid-Resistant Th17/Tc-17 Cells to TLR-2 and TLR-4 Ligands is a Feature of Multiple Sclerosis Patients at Higher Risk of Therapy Failure.

The presence of T cells expressing TLR-2 and TLR-4 has been associated with relapsing-remitting multiple sclerosis (RRMS) pathogenesis. Here, we evaluated whether the effectiveness of DMT in controlling clinical activity of the disease would be associated with modulation of proportion of TLRs+ T cells. Whole peripheral blood mononuclear cells, purified CD4+ and CD8+ T cells from RRMS patients were cultured with different stimuli. The frequency of IL-17-secreting CD4+ and CD8+ T cells positive for TLR-2 and TLR-4 was determined by flow cytometry. The cytokine profile of these T cells following TLR-2 and TLR-4 stimulation was determined by Multiplex. Some of these T cell cultures were treated with hydrocortisone. The levels of LPS-binding protein (LBP) were dosed by ELISA. Clinical (occurrence of relapses) and radiological (number of active brain lesions) activity were evaluated during the 1-year follow-up. Despite DMT, high intensity of TLR-2 and TLR-4 expression on (CD4+ and CD8+) T-cells, as well as the frequency of IL-17-secreting (CD4+ and CD8+) T-cells, are predictive of future RRMS relapses. Moreover, higher cytokine production related to Th17/Tc-17 phenotypes in response to TLR-2 and TLR-4 agonists was observed in DMT-treated patients and displayed an elevated number of brain lesions. The hyperresponsiveness of MS-derived T-cells to TLR-2 and TLR-4 ligands, with high levels of IL-1β, IL-6, IL-17, IFN-γ and GM-CSF in response to both TLR agonists, positively correlated with plasma LBP levels. Interestingly, corticoid was less efficient in reducing Th17 and Tc-17 cytokine production induced by TLR-2 and TLR-4 ligands in DMT-treated patients who relapsed during follow-up. Collectively, the data suggested that persistence of circulating Th17 and Tc17 cells expressing elevated levels of functional TLR-2 and TLR-4 could indicate high disease activity and lower therapeutic efficacy in RRMS patients.

Possible involvement of Toll-like receptor 8-positive monocytes/macrophages in the pathogenesis of Sjögren's disease.

Sjögren's disease (SjD) is an autoimmune disease marked by lymphocytic infiltration of salivary and lacrimal glands, leading to glandular dysfunction, where CD4-positive helper T (Th) cells and their cytokines are crucial in the pathogenesis. Recent studies have demonstrated that Toll-like receptors (TLRs), particularly those recognizing immune complexes containing DNA and RNA, contribute to Th cell activation in various autoimmune diseases. This study explores the expression and function of these TLRs in SjD. DNA microarray analysis of salivary gland tissue from six SjD patients and real-time PCR (n = 32) was used to identify overexpressed TLRs. Single-cell RNA sequencing (scRNA-seq) was performed using tissue lesions and integrated with published scRNA-seq data from tissues and peripheral blood mononuclear cells to examine gene expression in macrophages and monocytes. Finally, multi-color immunofluorescence staining was conducted to confirm TLR8 expression and function in SjD lesions (n = 19). DNA microarray analysis revealed the up-regulation of TLR8, along with other TLRs and innate immune response genes in SjD. Real-time PCR showed significant up-regulation of TLR7 and TLR8. TLR8 up-regulated in both analyses. In scRNA-seq analysis, the TLR8-expressing cluster comprised macrophages and monocytes, which also produced T cell activation genes like CD86. TLR8-positive macrophages infiltrated inflammatory sites and frequently expressed CD86 in quantitative imaging approaches. These results suggest that infiltrating monocytes and macrophages may produce cytokines and chemokines through TLR8 stimulation, potentially enhancing B7 molecule expression, promoting the adaptive immune response, and contributing to SjD pathogenesis.

Toll-Like Receptor 2 Attenuates the Formation and Progression of Angiotensin II-Induced Abdominal Aortic Aneurysm in ApoE−/− Mice

Introduction: We demonstrated Toll-like receptor (TLR) 4 in the pathogenesis of angiotensin II (AngII)-mediated abdominal aortic aneurysm (AAA) formation. Here, we study TLR2 in the AAA formation. Methods: Male ApoE−/− and ApoE−/−TLR2−/− mice were treated with AngII. Mice were injected with the TLR2 agonist Pam3CSK4. The incidence and severity of AAA were determined. MCP-1, MCP-5, RANTES, CXCL10, CCR5, and CXCR3 were analyzed. M1 and M2 macrophages in the aorta were detected by flow cytometry. Results: These studies demonstrated an increase in AAA formation in TLR2−/− mice and a decrease by Pam3CSK4. Pam3CSK4 decreased the ratio of M1/M2 and the levels of RANTES, CXCL10, CCR5, and CXCR3. Furthermore, Pam3CSK4 treatment 1 week following AngII retarded the progression of AAA. Conclusion: These data demonstrated a protective effect of TLR2 signaling on AAA in association with a decrease in the ratio of M1 to M2 macrophages and the expression of chemokines and their receptors. Furthermore, the treatment of Pam3CSK4 after AngII demonstrated a marked retardation of lesion progression. Given the fact that most AAA patients are detected late in the disease process, these findings suggest that TLR2 stimulation may play a therapeutic role in retarding disease progression.

Toll-like receptor 2-mediated ERK activation significantly upregulates interleukin-6 expression in M2-polarized macrophages

Abstract Objectives M2-polarized macrophages and interleukin (IL)-6 significantly alter the tumor microenvironment and promote the malignant behaviors of tumor cells. This study aimed to establish M2-type macrophages from THP-1 cells, which are human leukemia monocytes, and investigate the significance of toll-like receptor 2 (TLR2) signaling in IL-6 production. Methods THP-1 cells were treated with phorbol 12-myristate 13-acetate, IL-4, and IL-13 to stimulate their differentiation into M2 macrophages. Cell differentiation was confirmed by cytokine production, marker expression, and morphological alterations. Treatment with TLR agonists induced TLR stimulation in M2 macrophages. Subsequently, secretion and expression levels of IL-6 in M2 macrophages were measured using enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, and western blotting. Results Myeloid differentiation factor 88, tumor necrosis factor-associated factor 6, and IL-1 receptor-associated kinase-1/4 signaling pathways contributed to IL-6 production upon TLR2 activation in M2 macrophages. While both TLR2 and TLR4 activated NF-κB in M2 macrophages, IL-6 production was mainly dependent on TLR2, not TLR4, suggesting the involvement of major mechanisms other than NF-κB in IL-6 production. Notably, TLR2-stimulated extracellular signal-regulated kinase (ERK) was necessary for abundant IL-6 production, indicating that TLR2-mediated ERK signaling plays an essential role in M2 macrophages. Conclusions These results highlight the significance of TLR2 signaling in IL-6 production by M2 macrophages and provide insights into the underlying regulatory mechanisms.

Bacterial RNA sensing by TLR8 requires RNase 6 processing and is inhibited by RNA 2’O-methylation

TLR8 senses single-stranded RNA (ssRNA) fragments, processed via cleavage by ribonuclease (RNase) T2 and RNase A family members. Processing by these RNases releases uridines and purine-terminated residues resulting in TLR8 activation. Monocytes show high expression of RNase 6, yet this RNase has not been analyzed for its physiological contribution to the recognition of bacterial RNA by TLR8. Here, we show a role for RNase 6 in TLR8 activation. BLaER1 cells, transdifferentiated into monocyte-like cells, as well as primary monocytes deficient for RNASE6 show a dampened TLR8-dependent response upon stimulation with isolated bacterial RNA (bRNA) and also upon infection with live bacteria. Pretreatment of bacterial RNA with recombinant RNase 6 generates fragments that induce TLR8 stimulation in RNase 6 knockout cells. 2’O-RNA methyl modification, when introduced at the first uridine in the UA dinucleotide, impairs processing by RNase 6 and dampens TLR8 stimulation. In summary, our data show that RNase 6 processes bacterial RNA and generates uridine-terminated breakdown products that activate TLR8.

Suppressive effects of Toll-Like Receptor 2, Toll-Like Receptor 4, and Toll-Like Receptor 7 on protective responses to Mycobacterium bovis BCG from epithelial cells

Mycobacteria have several mechanisms for evasion of protective responses mounted by the host. In this study, we unravel yet another mechanism that is mediated by Toll-Like Receptors TLR2, TLR4, and TLR7 in epithelial cells. We show that mycobacterial infection of epithelial cells increases the expression of TLR2, TLR4, and TLR7. Stimulation of either TLR along with mycobacterial infection results in an inhibition of oxidative burst resulting in increased survival of mycobacteria inside epithelial cells. TLR stimulation along with mycobacterial infection also inhibits activation of epithelial cells for T cell responses by differentially regulating the activation of ERK-MAPK and p38-MAPK along with inhibition of co-stimulatory molecule CD86 expression. Furthermore, stimulation of either TLR inhibits the induction of apoptosis and autophagy. Knockdown of either TLR by specific siRNAs reverses the inhibition by ROS and apoptosis by mycobacteria and results in reduced intracellular survival of mycobacteria in a MyD88-dependent manner. These results point towards a negative role for TLR2, TLR4, and TLR7 in regulating protective responses to M. bovis BCG infection in epithelial cells.

Elevated levels of damage-associated molecular patterns HMGB1 and S100A8/A9 coupled with toll-like receptor-triggered monocyte activation are associated with inflammation in patients with myelofibrosis.

Inflammation plays a pivotal role in the pathogenesis of primary and post-essential thrombocythemia or post-polycythemia vera myelofibrosis (MF) in close cooperation with the underlying molecular drivers. This inflammatory state is induced by a dynamic spectrum of inflammatory cytokines, although recent evidence points to the participation of additional soluble inflammatory mediators. Damage-associated molecular patterns (DAMPs) represent endogenous signals released upon cell death or damage which trigger a potent innate immune response. We assessed the contribution of two prototypical DAMPs, HMGB1 and S100A8/A9, to MF inflammation. Circulating HMGB1 and S100A8/A9 were elevated in MF patients in parallel to the degree of systemic inflammation and levels increased progressively during advanced disease stages. Patients with elevated DAMPs had higher frequency of adverse clinical features, such as anemia, and inferior survival, suggesting their contribution to disease progression. Monocytes, which are key players in MF inflammation, were identified as a source of S100A8/A9 but not HMGB1 release, while both DAMPs correlated with cell death parameters, such as serum LDH and cell-free DNA, indicating that passive release is an additional mechanism leading to increased DAMPs. HMGB1 and S100A8/A9 promote inflammation through binding to Toll-like receptor (TLR) 4, whereas the former also binds TLR2. Monocytes from MF patients were shown to be hyperactivated at baseline, as reflected by higher CD11b and tissue factor exposure and increased expression levels of proinflammatory cytokines IL-1β and IL-6. Patient monocytes showed preserved TLR4 and TLR2 expression and were able to mount normal or even exacerbated functional responses and cytokine upregulation following stimulation of TLR4 and TLR2. Elevated levels of endogenous TLR ligands HMGB1 and S100A8/A9 coupled to the finding of preserved or hyperreactive TLR-triggered responses indicate that DAMPs may promote monocyte activation and cytokine production in MF, fueling inflammation. Plasma IL-1β and IL-6 were elevated in MF and correlated with DAMPs levels, raising the possibility that DAMPs could contribute to cytokine generation in vivo. In conclusion, this study highlights that, in cooperation with classic proinflammatory cytokines, DAMPs represent additional inflammatory mediators that may participate in the generation of MF inflammatory state, potentially providing novel biomarkers of disease progression and new therapeutic targets.

Role of lamin A/C on dendritic cell function in antiviral immunity

Dendritic cells (DCs) play a crucial role in orchestrating immune responses, particularly in promoting IFNγ-producing-CD8 cytotoxic T lymphocytes (CTLs) and IFNγ-producing-CD4 T helper 1 (Th1) cells, which are essential for defending against viral infections. Additionally, the nuclear envelope protein lamin A/C has been implicated in T cell immunity. Nevertheless, the intricate interplay between innate and adaptive immunity in response to viral infections, particularly the role of lamin A/C in DC functions within this context, remains poorly understood. In this study, we demonstrate that mice lacking lamin A/C in myeloid LysM promoter-expressing cells exhibit a reduced capacity to induce Th1 and CD8 CTL responses, leading to impaired clearance of acute primary Vaccinia virus (VACV) infection. Remarkably, in vitro-generated granulocyte macrophage colony-stimulating factor bone marrow-derived DCs (GM-CSF BMDCs) show high levels of lamin A/C. Lamin A/C absence on GM-CSF BMDCs does not affect the expression of costimulatory molecules on the cell membrane but it reduces the cellular ability to form immunological synapses with naïve CD4 T cells. Lamin A/C deletion induces alterations in NFκB nuclear localization, thereby influencing NF-κB-dependent transcription. Furthermore, lamin A/C ablation modifies the gene accessibility of BMDCs, predisposing these cells to mount a less effective antiviral response upon TLR stimulation. This study highlights the critical role of DCs in interacting with CD4 T cells during antiviral responses and proposes some mechanisms through which lamin A/C may modulate DC function via gene accessibility and transcriptional regulation.

Upregulation of key factors of viral entry of corona- and influenza viruses upon TLR3-signaling in cells from the respiratory tract and clinical treatment options by 1,8-Cineol.

At the end of the 2019 coronavirus pandemic (COVID-19), highly contagious variants of coronaviruses had emerged. Together with influenza viruses, different variants of the coronavirus currently cause most colds and require appropriate drug treatment. We have investigated the expression of important factors for the replication of these viruses, namely transmembrane protease serine subtype 2 (TMPRSS2), neuropilin1 (NRP1), and angiotensin converting enzyme 2 (ACE2) or tumor necrosis factor-α (TNF-α) after toll like receptor-3 (TLR-3) stimulation using RT-qPCR and flow cytometry (FC) analysis. As model served primary fibroblasts derived from the lung and nasal cavity, as well as epidermal stem cells and fully matured respiratory epithelium. The stimulated cell cultures were treated with pharmaceuticals (Dexamethasone and Enzalutamide) and the outcome was compared with the phytomedicine 1,8-Cineol. The stimulation of TLR3 is sufficient to induce the expression of exactly those targets that were highly expressed in the corresponding culture type, specifically ACE2 and TMPRSS2 in respiratory epithelial stem cells and NRP1 in fibroblast cells. It seems this self-perpetuating cycle of infection-driven upregulation of key viral replication factors by the innate immune system represents an evolutionary advantage for viruses using these transcripts as viral replication factors. Likewise, to the standard pharmaceuticals with proven clinical efficiency, 1,8-Cineol was able to disrupt this self-perpetuating cycle. Considering the minor side effects and negligible pharmacological interactions with other drugs, it is conceivable that an adjuvant or combinatorial therapy with 1,8-Cineol for respiratory diseases caused by corona- or influenza viruses would be beneficial.

Butyrate and propionate are microbial danger signals that activate the NLRP3 inflammasome in human macrophages upon TLR stimulation

Short-chain fatty acids (SCFAs) are immunomodulatory compounds produced by the microbiome through dietary fiber fermentation. Although generally considered beneficial for gut health, patients suffering from inflammatory bowel disease (IBD) display poor tolerance to fiber-rich diets, suggesting that SCFAs may have contrary effects under inflammatory conditions. To investigate this, we examined the effect of SCFAs on human macrophages in the presence of Toll-like receptor (TLR) agonists. In contrast to anti-inflammatory effects under steady-state conditions, we found that butyrate and propionate activated the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome in the presence of TLR agonists. Mechanistically, these SCFAs prevented transcription of FLICE-like inhibitory protein (cFLIP) and interleukin-10 (IL-10) through histone deacetylase (HDAC) inhibition, triggering caspase-8-dependent NLRP3 inflammasome activation. SCFA-driven NLRP3 activation was potassium efflux independent and did not result in cell death but rather triggered hyperactivation and IL-1β release. Our findings demonstrate that butyrate and propionate are bacterially derived danger signals that regulate NLRP3 inflammasome activation through epigenetic modulation of the inflammatory response.

Nanoparticles targeting immune checkpoint protein VISTA induce potent antitumor immunity

BackgroundImmune checkpoint protein V-domain immunoglobulin suppressor of T cell activation (VISTA) controls antitumor immunity and is a valuable target for cancer immunotherapy. Previous mechanistic studies have indicated that VISTA impairs...

Toll-Like Receptor 8 is Expressed in Monocytes in Contrast to Plasmacytoid Dendritic Cells and Mediates Aberrant Interleukin-10 Responses in Patients With Systemic Sclerosis.

Systemic sclerosis (SSc) is a severe rheumatic disease causing fibrotic tissue rearrangement. Aberrant toll-like receptor (TLR) 8 transcripts in plasmacytoid dendritic cells (pDCs) were recently linked to SSc pathogenesis, which is at least partially mediated by increased type I interferon (IFN-I) responses. Here, we addressed the functional role of TLR8 signaling in different immune cell subsets of patients with SSc. Monocytes, conventional dendritic cells (cDCs), and pDCs from the blood and skin of patients with SSc were analyzed for TLR8 protein expression. To assess TLR function, cytokine responses upon TLR7 and TLR8 stimulation were studied. To identify relevant alterations specific for patients with SSc (n = 16), patients with primary Sjögren disease (pSS; n = 10) and healthy controls (HCs; n = 13) were included into the study. In all individuals, TLR8 was expressed in monocytes and cDCs but not in pDCs. The TLR8 expression levels were overall similar in patients with SSc and pSS and HCs. Additionally, in all study participants, TLR8 stimulation of pDCs did not induce IFN-I expression. In contrast, monocytes from patients with SSc revealed increased interleukin (IL)-10 responses upon TLR8 (patients with SSc vs HCs, P = 0.0126) and TLR7/8 stimulation (patients with SSc vs HCs, P = 0.0170). TLR8 protein is not expressed in pDCs of patients with SSc. Accordingly, they do not respond to TLR8 stimulation. In contrast, monocytes of patients with SSc respond to TLR8 stimulation with increased IL-10 responses. Therefore, TLR8 signaling in monocytes participates in SSc pathogenesis by conferring aberrant IL-10 expression.

Mitochondria Activity and CXCR4 Collaboratively Promote the Differentiation of CD11c+ B Cells Induced by TLR9 in Lupus.

Lupus is characterized by the autoantibodies against nuclear Ags, underscoring the importance of identifying the B cell subsets driving autoimmunity. Our research focused on the mitochondrial activity and CXCR4 expression in CD11c+ B cells from lupus patients after ex vivo stimulation with a TLR9 agonist, CpG-oligodeoxyribonucleotide (ODN). We also evaluated the response of CD11c+ B cells in ODN-injected mice. Post-ex vivo ODN stimulation, we observed an increase in the proportion of CD11chi cells, with elevated mitochondrial activity and CXCR4 expression in CD11c+ B cells from lupus patients. In vivo experiments showed similar patterns, with TLR9 stimulation enhancing mitochondrial and CXCR4 activities in CD11chi B cells, leading to the generation of anti-dsDNA plasmablasts. The CXCR4 inhibitor AMD3100 and the mitochondrial complex I inhibitor IM156 significantly reduced the proportion of CD11c+ B cells and autoreactive plasmablasts. These results underscore the pivotal roles of mitochondria and CXCR4 in the production of autoreactive plasmablasts.

Cell-Type-Specific Effect of Innate Immune Signaling on Stress Granules

Stress granules (SGs) are cytoplasmic membraneless compartments that can form in stressed cells. There is an intricate relationship between SGs and innate immune signaling pathways. A previous study reported that the innate immune signaling mediated by Toll-like receptors (TLRs) can inhibit SGs induced by endoplasmic reticulum stress (ER stress) in bone-marrow-derived macrophages (BMDMs) and the chemotherapy drug oxaliplatin in B16 melanoma cells. We wanted to test if this observation can be generalized to other cell types. First, we recapitulated the results from the previous study showing TLR signaling-mediated inhibition of SGs in BMDMs induced by ER stress. However, SGs formed in response to ER stress were either not inhibited or only very weakly inhibited by TLR4 stimulation in human lung cancer-derived A549 cells, murine immortalized mouse lung fibroblasts (iMLFs) and primary murine mouse lung fibroblasts. This correlated with a weak induction of IKK complex kinase activity by TLR4 stimulation in these cells. SGs formed by sodium arsenite treatment also remained unaffected by TLR4 signaling. Our results indicate that the innate immune signaling-mediated inhibition of SGs is cell-type-dependent, thus opening a new avenue for mechanistic studies of the crosstalk between innate immune and stress signaling pathways.

The Aconitate Decarboxylase 1/Itaconate Pathway Modulates Immune Dysregulation and Associates with Cardiovascular Disease Markers and Disease Activity in Systemic Lupus Erythematosus.

The Krebs cycle enzyme aconitate decarboxylase 1 (ACOD1) mediates itaconate synthesis in monocytes and macrophages. Previously, we reported that administration of 4-octyl itaconate to lupus-prone mice abrogated immune dysregulation and clinical features. In this study, we explore the role of the endogenous ACOD1/itaconate pathway in the development of TLR7-induced lupus (imiquimod [IMQ] model). We found that, invitro, ACOD1 was induced in mouse bone marrow-derived macrophages and human monocyte-derived macrophages following TLR7 stimulation. This induction was partially dependent on type I IFN receptor signaling and on specific intracellular pathways. In the IMQ-induced mouse model of lupus, ACOD1 knockout (Acod1-/-) displayed disruptions of the splenic architecture, increased serum levels of anti-dsDNA and proinflammatory cytokines, and enhanced kidney immune complex deposition and proteinuria, when compared with the IMQ-treated wild-type mice. Consistent with these results, Acod1-/- bone marrow-derived macrophages treated invitro with IMQ showed higher proinflammatory features. Furthermore, itaconate serum levels in systemic lupus erythematosus patients were decreased compared with healthy individuals, in association with disease activity and specific perturbed cardiometabolic parameters. These findings suggest that the ACOD1/itaconate pathway plays important immunomodulatory and vasculoprotective roles in systemic lupus erythematosus, supporting the potential therapeutic role of itaconate analogs in autoimmune diseases.

Deleterious intestinal inflammation in neonatal mice treated with TLR2/TLR6 agonists.

By providing innate immune modulatory stimuli, the early-life immune system can be enhanced to increase resistance to infections. Activation of innate cell surface receptors called pattern recognition receptors by Toll-like receptor (TLR) ligands is one promising approach that can help to control infections as described for listeriosis and cryptosporidiosis. In this study, the effect of TLR2/TLR1 and TLR2/TLR6 agonists was compared when injected into neonatal mice. Surprisingly, the stimulation of TLR2/TLR6 led to the death of the neonatal mice, which was not observed in adult mice. The TLR2/TLR6 agonist administration induced higher systemic and intestinal inflammation in both adult and neonatal mice when compared with TLR2/TLR1 agonist. The mortality of neonatal mice was interferon γ dependent and involved the intestinal production of interleukin-22 and interleukin-17A. This study clearly demonstrates that targeting TLRs as new control strategy of neonatal infections has to be used with caution. Depending on its heterodimeric form, TLR2 stimulation can induce more or less severe adverse effects relying on the age-related immune functions of the host.