Validation of mitochondrial biomarkers and immune dynamics in polycystic ovary syndrome.

PROBLEM: Polycystic ovary syndrome (PCOS), a prevalent endocrine-metabolic disorder, presents considerable therapeutic challenges due to its complex and elusive pathophysiology. METHOD OF STUDY: We employed three machine learning algorithms to identify potential biomarkers within a training dataset, comprising GSE138518, GSE155489, and GSE193123. The diagnostic accuracy of these biomarkers was rigorously evaluated using a validation dataset using area under the curve (AUC) metrics. Further validation in clinical samples was conducted using PCR and immunofluorescence techniques. Additionally, we investigate the complex interplay among immune cells in PCOS using CIBERSORT to uncover the relationships between the identified biomarkers and various immune cell types. RESULTS: Our analysis identified ACSS2, LPIN1, and NR4A1 as key mitochondria-related biomarkers associated with PCOS. A notable difference was observed in the immune microenvironment between PCOS patients and healthy controls. In particular, LPIN1 exhibited a positive correlation with resting mast cells, whereas NR4A1 demonstrated a negative correlation with monocytes in PCOS patients. CONCLUSION: ACSS2, LPIN1, and NR4A1 emerge as PCOS-related diagnostic biomarkers and potential intervention targets, opening new avenues for the diagnosis and management of PCOS.

Characterization of the genetic determinants of context-specific DNA methylation in primary monocytes.

To better understand inter-individual variation in sensitivity of DNA methylation (DNAm) to immune activity, we characterized effects of inflammatory stimuli on primary monocyte DNAm (n = 190). We find that monocyte DNAm is site-dependently sensitive to lipopolysaccharide (LPS), with LPS-induced demethylation occurring following hydroxymethylation. We identify 7,359 high-confidence immune-modulated CpGs (imCpGs) that differ in genomic localization and transcription factor usage according to whether they represent a gain or loss in DNAm. Demethylated imCpGs are profoundly enriched for enhancers and colocalize to genes enriched for disease associations, especially cancer. DNAm is age associated, and we find that 24-h LPS exposure triggers approximately 6 months of gain in epigenetic age, directly linking epigenetic aging with innate immune activity. By integrating LPS-induced changes in DNAm with genetic variation, we identify 234 imCpGs under local genetic control. Exploring shared causal loci between LPS-induced DNAm responses and human disease traits highlights examples of disease-associated loci that modulate imCpG formation.

Repeated social defeat stress differently affects arthritis-associated hypersensitivity in male and female mice.

Chronic stress enhances the risk of neuropsychiatric disorders and contributes to the aggravation and chronicity of pain. The development of stress-associated diseases, including pain, is affected by individual vulnerability or resilience to stress, although the mechanisms remain elusive. We used the repeated social defeat stress model promoting susceptible and resilient phenotypes in male and female mice and induced knee mono-arthritis to investigate the impact of stress vulnerability on pain and immune system regulation. We analyzed different pain-related behaviors, measured blood cytokine and immune cell levels, and performed histological analyses at the knee joints and pain/stress-related brain areas. Stress susceptible male and female mice showed prolonged arthritis-associated hypersensitivity. Interestingly, hypersensitivity was exacerbated in male but not female mice. In males, stress promoted transiently increased neutrophils and Ly6Chigh monocytes, lasting longer in susceptible than resilient mice. While resilient male mice displayed persistently increased levels of the anti-inflammatory interleukin (IL)-10, susceptible mice showed increased levels of the pro-inflammatory IL-6 at the early- and IL-12 at the late arthritis stage. Although joint inflammation levels were comparable among groups, macrophage and neutrophil infiltration was higher in the synovium of susceptible mice. Notably, only susceptible male mice, but not females, presented microgliosis and monocyte infiltration in the prefrontal cortex at the late arthritis stage. Blood Ly6Chigh monocyte depletion during the early inflammatory phase abrogated late-stage hypersensitivity and the associated histological alterations in susceptible male mice. Thus, recruitment of blood Ly6Chigh monocytes during the early arthritis phase might be a key factor mediating the persistence of arthritis pain in susceptible male mice. Alternative neuro-immune pathways that remain to be explored might be involved in females.

Bone marrow CD8+ Trm cells induced by IL-15 and CD16+ monocytes contribute to HSPC destruction in human severe aplastic anemia.

Idiopathic severe aplastic anemia (SAA) is a disease of bone marrow failure caused by T-cell-induced destruction of hematopoietic stem and progenitor cells (HSPCs), however the mechanism remains unclear. We performed single-cell RNA sequencing of PBMCs and BMMCs from SAA patients and healthy donors and identified a CD8+ T cell subset with a tissue residency phenotype (Trm) in bone marrow that exhibit high IFN-γ and FasL expression and have a higher ability to induce apoptosis in HSPCs in vitro through FasL expression. CD8+ Trm cells were induced by IL-15 presented by IL-15Rα on monocytes, especially CD16+ monocytes, which were increased in SAA patients. CD16+ monocytes contributed to IL-15-induced CD38+CXCR6+ pre-Trm differentiation into CD8+ Trm cells, which can be inhibited by the CD38 inhibitor 78c. Our results demonstrate that IL-15-induced CD8+ Trm cells are pathogenic cells that mediate HSPC destruction in SAA patients and are therapeutic targets for future treatments.

Polymer Backpack-Loaded Tissue Infiltrating Monocytes for Treating Cancer.

Adoptive cell therapies are dramatically altering the treatment landscape of cancer. However, treatment of solid tumors remains a major unmet need, in part due to limited adoptive cell infiltration into the tumor and in part due to the immunosuppressive tumor microenvironment. The heterogeneity of tumors and presence of nonresponders also call for development of antigen-independent therapeutic approaches. Myeloid cells offer such an opportunity, given their large presence in the immunosuppressive tumor microenvironment, such as in triple negative breast cancer. However, their therapeutic utility is hindered by their phenotypic plasticity. Here, the impressive trafficking ability of adoptively transferred monocytes is leveraged into the immunosuppressive 4T1 tumor to develop an antitumor therapy. To control monocyte differentiation in the tumor microenvironment, surface-adherent "backpacks" stably modified with interferon gamma (IFNγ) are developed to stimulate macrophage plasticity into a pro-inflammatory, antitumor phenotype, a strategy as referred to as Ornate Polymer backpacks on Tissue Infiltrating Monocytes (OPTIMs). Treatment with OPTIMs substantially reduces tumor burden in a mouse 4T1 model and significantly increases survival. Cytokine and immune cell profiling reveal that OPTIMs remodeled the tumor microenvironment into a pro-inflammatory state.

Truncating NFKB1 variants cause combined NLRP3 inflammasome activation and type I interferon signaling and predispose to necrotizing fasciitis.

In monogenic autoinflammatory diseases, mutations in genes regulating innate immune responses often lead to uncontrolled activation of inflammasome pathways or the type I interferon (IFN-I) response. We describe a mechanism of autoinflammation potentially predisposing patients to life-threatening necrotizing soft tissue inflammation. Six unrelated families are identified in which affected members present with necrotizing fasciitis or severe soft tissue inflammations. Exome sequencing reveals truncating monoallelic loss-of-function variants of nuclear factor κ light-chain enhancer of activated B cells (NFKB1) in affected patients. In patients' macrophages and in NFKB1-variant-bearing THP-1 cells, activation increases both interleukin (IL)-1ß secretion and IFN-I signaling. Truncation of NF-κB1 impairs autophagy, accompanied by the accumulation of reactive oxygen species and reduced degradation of inflammasome receptor nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein 3 (NLRP3), and Toll/IL-1 receptor domain-containing adaptor protein inducing IFN-ß (TRIF), thus leading to combined excessive inflammasome and IFN-I activity. Many of the patients respond to anti-inflammatory treatment, and targeting IL-1ß and/or IFN-I signaling could represent a therapeutic approach for these patients.

Moesin deficiency leads to lupus-like nephritis with accumulation of CXCL13-producing patrolling monocytes.

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that link plasma membrane proteins to the cortical cytoskeleton and thus regulate diverse cellular processes. Mutations in the human moesin gene cause a primary immunodeficiency called X-linked moesin-associated immunodeficiency (X-MAID), which may be complicated by an autoimmune phenotype with kidney involvement. We previously reported that moesin-deficient mice exhibit lymphopenia similar to that of X-MAID and develop a lupus-like autoimmune phenotype with age. However, the mechanism through which moesin defects cause kidney pathology remains obscure. Here, we characterized immune cell infiltration and chemokine expression in the kidney of moesin-deficient mice. We found accumulation of CD4+ T and CD11b+ myeloid cells and high expression of CXCL13, whose upregulation was detected before the onset of overt nephritis. CD4+ T cell population contained IFN-γ-producing effectors and expressed the CXCL13 receptor CXCR5. Among myeloid cells, Ly6Clo patrolling monocytes and MHCIIlo macrophages markedly accumulated in moesin-deficient kidneys and expressed high CXCL13 levels, implicating the CXCL13-CXCR5 axis in nephritis development. Functionally, Ly6Clo monocytes from moesin-deficient mice showed reduced migration toward sphingosine 1-phosphate. These findings suggest that moesin plays a role in regulating patrolling monocyte homeostasis, and that its defects lead to nephritis associated with accumulation of CXCL13-producing monocytes and macrophages.

Effect of cholecalciferol supplementation on hand grip strength, walking speed, and expression of vitamin D receptor, interleukin-6, and insulin-like growth factor-1 in monocyte in pre-frail older adults: A randomized double-blind placebo-controlled trial.

AIM: To investigate the effect of cholecalciferol supplementation on hand grip strength, walking speed, and expression of vitamin D receptor (VDR), interleukine-6 (IL-6) and insulin-like growth factor-1 (IGF-1) in monocyte in pre-frail older adults. METHODS: We conducted a randomized double-blinded placebo-controlled clinical trial for 12 weeks, involving 120 pre-frail older adults who were randomized to the cholecalciferol group (cholecalciferol 4000 IU/day) or the placebo group. All subjects were given calcium lactate 500 mg/day. Hand grip strength and walking speed, as primary outcomes, were analyzed using intention-to-treat analysis. The expression of VDR, IGF-1 and IL-6 in monocytes, as secondary outcomes, were analyzed using per-protocol analysis. RESULTS: After a 12-week intervention, there was a significant increase in serum 25(OH)D levels in both groups, with the increase being higher in the cholecalciferol group than in the placebo group (49.05 vs. 24.01 ng/mL; P < 0.001). No statistically significant differences were observed in hand grip strength (P = 0.228) and walking speed (P = 0.734) between the groups. There were no differences in the expression of VDR (P = 0.513), IL-6 (P = 0.509), and IGF-1 (P = 0.503) monocytes between the groups. CONCLUSIONS: Cholecalciferol supplementation for 12 weeks increased serum 25(OH)D levels among pre-frail older adults. However, it did not improve hand grip strength and walking speed, and nor did it change the expression of VDR, IL-6, and IGF-1 in monocytes. Geriatr Gerontol Int 2024; ••: ••-••.

Studying Monocyte-Myofibroblast Immunomodulation in a 3D Co-culture System.

Simple and reproducible 3D cell culture systems that mimic biological interactions within physiological tissues (biomimetics) can provide unique insight for scientific inquiries compared to 2D cell cultures. Fibroblast-populated collagen lattices (FPCLs) are commonly used for mimicking physiological collagen matrices, potentiating biomechanical stresses on embedded fibroblasts. Here, we describe a novel 3D co-culture model that incorporates human Tenon's capsule fibroblasts embedded in FPCLs co-cultured with THP-1 monocytes suspended in culture media. This method can be used for the assessment of cell-cell interactions in various stages of the wound healing process and can facilitate various types of immune cells in co-culture. This system can also be used to study pharmacological agents that may eventually improve clinical outcomes in patients affected by inflammatory disorders.

Mimicking the cellular environment does not cause monocyte-derived macrophages to become phenotypically similar to Kupffer cells.

Resident macrophages of various mammalian organs are characterized by several distinctive features in their gene expression profile and phenotype, including involvement in the regulation of organ functions, as well as reduced sensitivity to proinflammatory activation factors. The reasons for the formation of such a specific phenotype remain the subject of intensive research. Some papers emphasize the role of the origin of organ macrophages. Other studies indicate that monocytes that develop in the red bone marrow are also able to form resident macrophages with a phenotype characteristic of a particular organ, but this requires appropriate microenvironmental conditions. In this article, we studied the possibility of differentiation of monocyte-derived macrophages into cells with a Kupffer-like phenotype under the influence of the main stromal components of Kupffer cells macrophage niche: Ito cells, liver sinusoid endotheliocytes and hepatocyte growth factor (HGF). It was found that Kupffer cells are characterized by several features, including increased expression of transcription factors Spic and Id3, as well as MUP family genes, Clusterin and Ngp genes. In addition, Kupffer cells were characterized by a higher proliferative activity. The expression of marker genes of Kupffer cells (i.e. Id3, Spic, Marco and Timd4) increased in monocyte-derived macrophages during coculture with Ito cells, liver sinusoid endothelial cells, macrophage colony-stimulating factor and HGF cells only by 3 days. However, the expression level of these genes was always higher in Kupffer cells. In addition, a complete coincidence of the expressed gene profile in monocyte-derived macrophages and Kupffer cells did not occur even after 3 days of culturing.

The monocyte cell surface is a unique site of autoantigen generation in rheumatoid arthritis.

Although anti-citrullinated protein autoantibodies (ACPAs) are a hallmark serological feature of rheumatoid arthritis (RA), the mechanisms and cellular sources behind the generation of the RA citrullinome remain incompletely defined. Peptidylarginine deiminase IV (PAD4), one of the key enzymatic drivers of citrullination in the RA joint, is expressed by granulocytes and monocytes; however, the subcellular localization and contribution of monocyte-derived PAD4 to the generation of citrullinated autoantigens remain underexplored. In this study, we demonstrate that PAD4 displays a widespread cellular distribution in monocytes, including expression on the cell surface. Surface PAD4 was enzymatically active and capable of citrullinating extracellular fibrinogen and endogenous surface proteins in a calcium dose-dependent manner. Fibrinogen citrullinated by monocyte-surface PAD4 could be specifically recognized over native fibrinogen by a panel of eight human monoclonal ACPAs. Several unique PAD4 substrates were identified on the monocyte surface via mass spectrometry, with citrullination of the CD11b and CD18 components of the Mac-1 integrin complex being the most abundant. Citrullinated Mac-1 was found to be a target of ACPAs in 25% of RA patients, and Mac-1 ACPAs were significantly associated with HLA-DRB1 shared epitope alleles, higher C-reactive protein and IL-6 levels, and more erosive joint damage. Our findings implicate the monocyte cell surface as a unique and consequential site of extracellular and cell surface autoantigen generation in RA.

Sphingosine-1-Phosphate Shapes Healthy Monocytes into An Immunosuppressive Phenotype.

BACKGROUND/AIMS: The physiological phenotype of individuals can influence and shape real-life phenomena in that it can contribute to the development of specific characteristics that can affect the immune response to specific stimuli. In this study we aimed to understand whether the sphingosine/sphingosine-1-phoshate (S1P) axis can modulate the immunotype of circulating cells. METHODS: To pursue this goal, we performed bioinformatic analyses of public datasets. RESULTS: The transcriptomic profile of healthy subjects of GSE192829 dataset identified two clusters with different transcriptional repertoire. Cluster 1 expressed higher levels of enzymes for S1P formation than cluster 0 which was characterized by enzymes that lead to ceramide formation, which represent the opposite metabolic direction. Inference analysis showed that cluster 1 was higher populated by monocytes, CD4+ T and B cells than cluster 0. Of particular interest was the phenotype of the monocytes in cluster 1 which showed an immunosuppressive nature compared to those in cluster 0. The role of S1P signature in healthy PBMCs was confirmed with other dataset analyses, supporting that circulating monocytes positive to the ceramidase, unlike the negative ones, had an immunosuppressive phenotype characterized by hub immunosuppressive markers (i.e. TYROBP, FCER1G, SYK, SIRPA, CSF1R, AIF1, FCGR2A, CLEC7A, LYN, PLCG2, LILRs, HCK, GAB2). This hub genes well discriminated the immunotype of healthy subjects. CONCLUSION: In conclusion this study highlights that S1P-associated hub markers can be useful to discriminate subjects with pronounced immunosuppression.

Interferons and epigenetic mechanisms in training, priming and tolerance of monocytes and hematopoietic progenitors.

Training and priming of innate immune cells involve preconditioning by PAMPs, DAMPs, and/or cytokines that elicits stronger induction of inflammatory genes upon secondary challenge. Previous models distinguish training and priming based upon whether immune activation returns to baseline prior to secondary challenge. Tolerance is a protective mechanism whereby potent stimuli induce refractoriness to secondary challenge. Training and priming are important for innate memory responses that protect against infection, efficacy of vaccines, and maintaining innate immune cells in a state of readiness; tolerance prevents toxicity from excessive immune activation. Dysregulation of these processes can contribute to pathogenesis of autoimmune/inflammatory conditions, post-COVID-19 hyperinflammatory states, or sepsis-associated immunoparalysis. Training, priming, and tolerance regulate similar "signature" inflammatory genes such as TNF, IL6, and IL1B and utilize overlapping epigenetic mechanisms. We review how interferons (IFNs), best known for activating JAK-STAT signaling and interferon-stimulated genes, also play a key role in regulating training, priming, and tolerance via chromatin-mediated mechanisms. We present new data on how monocyte-to-macrophage differentiation modulates IFN-γ-mediated priming, affects regulation of AP-1 and CEBP activity, and attenuates superinduction of inflammatory genes. We present a "training-priming continuum" model that integrates IFN-mediated priming into current concepts about training and tolerance and proposes a central role for STAT1 and IRF1.

Characterization of blue-green blood leukocyte inclusions and accompanying clinical, hematologic, and serum biochemical changes in dogs.

BACKGROUND: Lipofuscin-like cytoplasmic inclusions have been reported in human blood neutrophils and monocytes but have not been described in dogs. In people, these "green granules of death" have been associated with moderate to severe hepatocellular injury and high mortality. OBJECTIVES: To describe clinicopathologic abnormalities, diagnoses, and outcomes of dogs with greenish inclusions in blood neutrophils or monocytes, and to determine if the inclusions have features of lipofuscin. METHODS: Clinical cases were identified prospectively through routine evaluation of CBC samples. Leukocyte inclusions were characterized with routine staining and assessed for iron and autofluorescence. Additional cases were identified by examination of archived blood smears from dogs meeting search criteria for hepatocellular injury, and clinicopathologic findings were recorded. RESULTS: All 7 prospectively identified dogs with inclusions had inflammation and moderate to marked increases in serum alanine aminotransferase (ALT) activity, as did the 4 dogs identified from the 97 meeting retrospective search criteria. The inclusions were Prussian blue-negative (5/5) with broad-spectrum autofluorescence (5/5) and the appearance of lipofuscin with and without Wright staining. Most clinical diagnoses involved hepatic disorders (5/7 prospective and 3/4 retrospective cases) or pancreatitis (3/7 prospective and 2/4 retrospective cases), and some involved both; 8 of 11 dogs died within 7 days of admission. CONCLUSIONS: Blue-green cytoplasmic inclusions uncommonly found in blood neutrophils ± monocytes of routine canine blood smears have stained and unstained properties of lipofuscin and suggest the presence of hepatocellular injury, often severe. Reporting these inclusions is recommended to guide clinical management.

Prognostic Roles of Inflammatory Biomarkers in Radioiodine-Refractory Thyroid Cancer Treated with Lenvatinib.

Background: Inflammatory biomarkers, such as the neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and platelet-to-lymphocyte ratio (PLR), serve as valuable prognostic indicators in various cancers. This multicenter, retrospective cohort study assessed the treatment outcomes of lenvatinib in 71 patients with radioactive iodine (RAI)-refractory thyroid cancer, considering the baseline inflammatory biomarkers. Methods: This study retrospectively included patients from five tertiary hospitals in Korea whose complete blood counts were available before lenvatinib treatment. Progression-free survival (PFS) and overall survival (OS) were evaluated based on the median value of inflammatory biomarkers. Results: No significant differences in baseline characteristics were observed among patients grouped according to the inflammatory biomarkers, except for older patients with a higher-than-median NLR (≥2) compared to their counterparts with a lower NLR (P= 0.01). Patients with a higher-than-median NLR had significantly shorter PFS (P=0.02) and OS (P=0.017) than those with a lower NLR. In multivariate analysis, a higher-than-median NLR was significantly associated with poor OS (hazard ratio, 3.0; 95% confidence interval, 1.24 to 7.29; P=0.015). However, neither the LMR nor the PLR was associated with PFS. A higher-than-median LMR (≥3.9) was significantly associated with prolonged OS compared to a lower LMR (P=0.036). In contrast, a higher-than-median PLR (≥142.1) was associated with shorter OS compared to a lower PLR (P=0.039). Conclusion: Baseline inflammatory biomarkers can serve as predictive indicators of PFS and OS in patients with RAI-refractory thyroid cancer treated with lenvatinib.

The association of TNF-alpha secretion and mtDNA copy number in CD14+ monocytes of patients with obesity and CHD.

Introduction: Mitochondrial dysfunction may be one of the causes of inflammatory activation of monocytes and macrophages, which leads to excessive secretion of inflammatory mediators and the development of chronic inflammation. Aims: The study was aimed to evaluate the secretion of inflammatory cytokine tumor necrosis factor-α (TNF-α) in the primary culture of monocytes, and to analyze its relationship with the number of mitochondrial DNA (mtDNA) copies in the blood of patients with coronary heart disease (CHD) and obesity. Materials and methods: 108 patients with obesity and concomitant CHD and a control group of 25 participants were included in the study. CD14+ monocytes were isolated by a standard method in a ficoll-urographin gradient, followed by separation using magnetic particles. The number of mtDNA copies was estimated using qPCR. Results: It was demonstrated that the number of mtDNA copies was significantly increased in groups of patients with CHD and obesity + CHD in comparison with control group. mtDNA copy number positively correlated with basal and LPS-stimulated TNF-α secretion, the most significant correlation was found in the group of patients with CHD and obesity. Conclusion: Thus, the change in mtDNA copy number in CD14+ monocytes which indicates the presence of mitochondrial dysfunction, confirm the direct involvement of mitochondria in the violation of the inflammatory response of monocytes revealed in this study as an increased secretion of inflammatory cytokine TNF-α.

Monocytic microRNAs-Novel targets in atherosclerosis therapy.

Atherosclerosis is a chronic proinflammatory disease of the vascular wall resulting in narrowing of arteries due to plaque formation, thereby causing reduced blood supply that is the leading cause for diverse end-organ damage with high mortality rates. Monocytes/macrophages, activated by elevated circulating lipoproteins, are significantly involved in the formation and development of atherosclerotic plaques. The imbalance between proinflammatory and anti-inflammatory macrophages, arising from dysregulated macrophage polarization, appears to be a driving force in this process. Proatherosclerotic processes acting on monocytes/macrophages include accumulation of cholesterol in macrophages leading to foam cell formation, as well as dysfunctional efferocytosis, all of which contribute to the formation of unstable plaques. In recent years, microRNAs (miRs) were identified as factors that could modulate monocyte/macrophage function and may therefore interfere with the atherosclerotic process. In this review, we present effects of monocyte/macrophage-derived miRs on atherosclerotic processes in order to reveal new treatment options using miRmimics or antagomiRs.

Tacrolimus alleviates pulmonary fibrosis progression through inhibiting the activation and interaction of ILC2 and monocytes.

Idiopathic pulmonary fibrosis (IPF) is a heterogeneous group of lung diseases with different etiologies and characterized by progressive fibrosis. This disease usually causes pulmonary structural remodeling and decreased pulmonary function. The median survival of IPF patients is 2-5 years. Predominantly accumulation of type II innate immune cells accelerates fibrosis progression by secreting multiple pro-fibrotic cytokines. Group 2 innate lymphoid cells (ILC2) and monocytes/macrophages play key roles in innate immunity and aggravate the formation of pro-fibrotic environment. As a potent immunosuppressant, tacrolimus has shown efficacy in alleviating the progression of pulmonary fibrosis. In this study, we found that tacrolimus is capable of suppressing ILC2 activation, monocyte differentiation and the interaction of these two cells. This effect further reduced activation of monocyte-derived macrophages (Mo-M), thus resulting in a decline of myofibroblast activation and collagen deposition. The combination of tacrolimus and nintedanib was more effective than either drug alone. This study will reveal the specific process of tacrolimus alleviating pulmonary fibrosis by regulating type II immunity, and explore the potential feasibility of tacrolimus combined with nintedanib in the treatment of pulmonary fibrosis. This project will provide new ideas for clinical optimization of anti-pulmonary fibrosis drug strategies.

Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells.

HIV-1 has a broad range of nuanced interactions with the immune system, and the incorporation of cellular proteins by nascent virions continues to redefine our understanding of the virus-host relationship. Proteins located at the sites of viral egress can be selectively incorporated into the HIV-1 envelope, imparting new functions and phenotypes onto virions, and impacting viral spread and disease. Using virion capture assays and western blot, we show that HIV-1 can incorporate the myeloid antigen CD14 into its viral envelope. Virion-incorporated CD14 remained biologically active and able to bind its natural ligand, bacterial lipopolysaccharide (LPS), as demonstrated by flow virometry and immunoprecipitation assays. Using a Toll-like receptor 4 (TLR4) reporter cell line, we also demonstrated that virions with bound LPS can trigger TLR4 signaling to activate transcription factors that regulate inflammatory gene expression. Complementary assays with THP-1 monocytes demonstrated enhanced secretion of inflammatory cytokines like tumor necrosis factor alpha (TNF-α) and the C-C chemokine ligand 5 (CCL5), when exposed to LPS-loaded virus. These data highlight a new type of interplay between HIV-1 and the myeloid cell compartment, a previously well-established cellular contributor to HIV-1 pathogenesis and inflammation. Persistent gut inflammation is a hallmark of chronic HIV-1 infection, and contributing to this effect is the translocation of microbes across the gut epithelium. Our data herein provide proof of principle that virion-incorporated CD14 could be a novel mechanism through which HIV-1 can drive chronic inflammation, facilitated by HIV-1 particles binding bacterial LPS and initiating inflammatory signaling in TLR4-expressing cells.IMPORTANCEHIV-1 establishes a lifelong infection accompanied by numerous immunological changes. Inflammation of the gut epithelia, exacerbated by the loss of mucosal T cells and cytokine dysregulation, persists during HIV-1 infection. Feeding back into this loop of inflammation is the translocation of intestinal microbes across the gut epithelia, resulting in the systemic dissemination of bacterial antigens, like lipopolysaccharide (LPS). Our group previously demonstrated that the LPS receptor, CD14, can be readily incorporated by HIV-1 particles, supporting previous clinical observations of viruses derived from patient plasma. We now show that CD14 can be incorporated by several primary HIV-1 isolates and that this virion-incorporated CD14 can remain functional, enabling HIV-1 to bind to LPS. This subsequently allowed CD14+ virions to transfer LPS to monocytic cells, eliciting pro-inflammatory signaling and cytokine secretion. We posit here that virion-incorporated CD14 is a potential contributor to the dysregulated immune responses present in the setting of HIV-1 infection.