Primary Monocyte-derived Macrophages Research Articles

Swertianin Promotes Anti-Tumor activity by facilitating Macrophage M1 polarization via STING signaling

To investigate the mechanism by which swertiamarin (swertianin, SWE) regulates the polarization of tumor microenvironment-associated macrophages to M1 phenotype, thereby exerting anti-tumor effects.SWE promoted the formation of M1 cells and increased the proportion of CD86 + cells in both RAW264.7 and primary monocyte-derived macrophages, while activating the STING-NF-κB pathway. When STING or P65 was knocked out, the effects of SWE were antagonized, inhibiting the formation of CD86 + M1 cells. At the animal level, SWE inhibited tumor growth, activated STING-NF-κB, and promoted the formation of CD86 + cells. STING-KO inhibited the effects of SWE.SWE can activate the STING-NF-κB signal to promote macrophage M1 polarization, playing an anti-tumor role.

SARS-CoV-2 Modulation of HIV Latency Reversal in a Myeloid Cell Line: Direct and Bystander Effects.

Coronavirus disease 2019 (COVID-19) might impact disease progression in people living with HIV (PLWH), including those on effective combination antiretroviral therapy (cART). These individuals often experience chronic conditions characterized by proviral latency or low-level viral replication in CD4+ memory T cells and tissue macrophages. Pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IFN-γ, can reactivate provirus expression in both primary cells and cell lines. These cytokines are often elevated in individuals infected with SARS-CoV-2, the virus causing COVID-19. However, it is still unknown whether SARS-CoV-2 can modulate HIV reactivation in infected cells. Here, we report that exposure of the chronically HIV-1-infected myeloid cell line U1 to two different SARS-CoV-2 viral isolates (ancestral and BA.5) reversed its latent state after 24 h. We also observed that SARS-CoV-2 exposure of human primary monocyte-derived macrophages (MDM) initially drove their polarization towards an M1 phenotype, which shifted towards M2 over time. This effect was associated with soluble factors released during the initial M1 polarization phase that reactivated HIV production in U1 cells, like MDM stimulated with the TLR agonist resiquimod. Our study suggests that SARS-CoV-2-induced systemic inflammation and interaction with macrophages could influence proviral HIV-1 latency in myeloid cells in PLWH.

Targeting tumor-associated macrophage-derived CD74 improves efficacy of neoadjuvant chemotherapy in combination with PD-1 blockade for cervical cancer

BackgroundCervical cancer has the second-highest mortality rate among malignant tumors of the female reproductive system. Immune checkpoint inhibitors such as programmed cell death protein 1 (PD-1) blockade are promising therapeutic...

Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses.

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.

A macrophage-cell model of HIV latency reveals the unusual importance of the bromodomain axis

BackgroundAlthough macrophages are now recognized as an essential part of the HIV latent reservoir, whether and how viral latency is established and reactivated in these cell types is poorly understood. To understand the fundamental mechanisms of viral latency in macrophages, there is an urgent need to develop latency models amenable to genetic manipulations and screening for appropriate latency-reversing agents (LRAs). Given that differentiated THP-1 cells resemble monocyte-derived macrophages in HIV replication mechanisms, we set out to establish a macrophage cell model for HIV latency using THP-1 cells.MethodsWe created single-cell clones of THP-1 cells infected with a single copy of the dual-labeled HIVGKO in which a codon switched eGFP (csGFP) is under the control of the HIV-1 5’ LTR promoter, and a monomeric Kusabira orange 2 (mKO2) under the control of cellular elongation factor one alpha promoter (EF1α). Latently infected cells are csGFP−, mKO2+, while cells with actively replicating HIV (or reactivated virus) are csGFP+,mKO2+. After sorting for latently infected cells, each of the THP-1 clones with unique integration sites for HIV was differentiated into macrophage-like cells with phorbol 12-myristate 13-acetate (PMA) and treated with established LRAs to stimulate HIV reactivation. Monocyte-derived macrophages (MDMs) harboring single copies of HIVGKO were used to confirm our findings.ResultsWe obtained clones of THP-1 cells with latently infected HIV with unique integration sites. When the differentiated THP-1 or primary MDMs cells were treated with various LRAs, the bromodomain inhibitors JQ1 and I-BET151 were the most potent compounds. Knockdown of BRD4, the target of JQ1, resulted in increased reactivation, thus confirming the pharmacological effect. The DYRK1A inhibitor Harmine and lipopolysaccharide (LPS) also showed significant reactivation across all three MDM donors. Remarkably, LRAs like PMA/ionomycin, bryostatin-1, and histone deacetylase inhibitors known to potently reactivate latent HIV in CD4 + T cells showed little activity in macrophages.ConclusionsOur results indicate that this model could be used to screen for appropriate LRAs for macrophages and show that HIV latency and reactivation mechanisms in macrophages may be distinct from those of CD4 + T cells.

Cell-intrinsic C5a synergizes with Dectin-1 in macrophages to mediate fungal killing

The complement factor C5a is a core effector product of complement activation. C5a, acting through its receptors C5aR1 and C5aR2, exerts pleiotropic immunomodulatory functions in myeloid cells, which is vital for host defense against pathogens. Pattern-recognition receptors (PRRs) are similarly expressed by immune cells as detectors of pathogen-associated molecular patterns. Although there is evidence of cross talk between complement and PRR signaling pathways, knowledge of the full potential for C5a-PRR interaction is limited. In this study, we comprehensively investigated how C5a signaling through C5a receptors can modulate diverse PRR-mediated cytokine responses in human primary monocyte-derived macrophages and observed a powerful, concentration-dependent bidirectional effect of C5a on PRR activities. Unexpectedly, C5a synergized with Dectin-1, Mincle, and STING in macrophages to a much greater extent than TLRs. Notably, we also identified that selective Dectin-1 activation using depleted zymosan triggered macrophages to generate cell-intrinsic C5a, which acted on intracellular and cell surface C5aR1, to help sustain mitochondrial ROS generation, up-regulate TNFα production, and enhance fungal killing. This study adds further evidence to the holistic functions of C5a as a central immunomodulator and important orchestrator of pathogen sensing and killing by phagocytes.

TOWARDS FUNCTIONAL PATIENT-DERIVED ORGANOIDS AS MODELS FOR SOFT-TISSUE JOINT DISEASE

In-vitro models of disease are valuable tools for studying disease and analysing response to therapeutics. Recently, advances in patient-derived organoid (PDO) models have been shown to faithfully recapitulate structure, function, and therapeutic response for a wide range of tissues. Frozen shoulder is a rare example of a chronic inflammatory fibrotic disease which is self-limiting, unlike many other soft tissue fibrotic disorders. As no in-vitro 3D models or in-vivo animal models exist for frozen shoulder, establishing an organoid model which recapitulates core diseases features may give insight into fibrosis resolution. Consequently, using biocompatible hydrogels, primary capsular fibroblasts, monocyte-derived macrophages and HUVEC cells, we generated stable PDO cultures which exhibited key disease phenotypes, including vascularization, increased stiffness, and an expanded lining layer over 21 days of culture. Through further investigation of cell-matrix and cell-cell interactions in the organoid model, we intend to unpack the differences between resolving and non-resolving fibrotic disease and uncover clinically relevant therapeutic targets for fibrosis.

Alkyne-tagged imidazolium-based membrane cholesterol analogs for Raman imaging applications.

Cholesterol is an important lipid playing a crucial role in mediating essential cellular processes as well as maintaining the basic structural integrity of biological membranes. Given its vast biological importance, there is an unabated need for sophisticated strategies to investigate cholesterol-mediated biological processes. Raman-tagged sterol analogs offer the advantage of being visualizable without the need for a bulky dye that potentially affects natural membrane integration and cellular interactions as it is the case for many conventionally used fluorescent analogs. Herein, we report a series of alkyne-tagged imidazolium-based cholesterol analogs (CHIMs) with large Raman scattering cross-sections that readily integrate into HEK cells and primary monocyte-derived macrophages and allow (multiplexed) cellular Raman imaging. We envision Raman-tagged CHIM analogs to be a powerful platform for the investigation of cholesterol-mediated cellular processes complementary to other established methods, such as the use of fluorescent analogs.

Alpha-synuclein shapes monocyte and macrophage cell biology and functions by bridging alterations of autophagy and inflammatory pathways.

Introduction: Abnormal spreading of alpha-synuclein (αS), a hallmark of Parkinson's disease, is known to promote peripheral inflammation, which occurs in part via functional alterations in monocytes/macrophages. However, underlying intracellular mechanisms remain unclear. Methods: Herein we investigate the subcellular, molecular, and functional effects of excess αS in human THP-1 monocytic cell line, THP-1-derived macrophages, and at least preliminarily, in primary monocyte-derived macrophages (MDMs). In cells cultured w/wo recombinant αS (1μM) for 4h and 24h, by Confocal microscopy, Western Blot, RT-qPCR, Elisa, and Flow Cytometry we assessed: i) αS internalization; ii) cytokine/chemokine expression/secretion, and C-C motif chemokine receptor 2 (CCR2) levels; iii) autophagy (LC3II/I, LAMP1/LysoTracker, p62, pS6/total S6); and iv) lipid droplets (LDs) accumulation, and cholesterol pathway gene expression. Transwell migration assay was employed to measure THP-1 cell migration/chemotaxis, while FITC-IgG-bead assay was used to analyze phagocytic capacity, and the fate of phagocytosed cargo in THP-1-derived macrophages. Results: Extracellular αS was internalized by THP-1 cells, THP-1-derived macrophages, and MDMs. In THP1 cells, αS induced a general pro-inflammatory profile and conditioned media from αS-exposed THP-1 cells potently attracted unstimulated cells. However, CCL2 secretion peaked at 4h αS, consistent with early internalization of its receptor CCR2, while this was blunted at 24h αS exposure, when CCR2 recycled back to the plasma membrane. Again, 4h αS-exposed THP-1 cells showed increased spontaneous migration, while 24h αS-exposed cells showed reduced chemotaxis. This occurred in the absence of cell toxicity and was associated with upregulation of autophagy/lysosomal markers, suggesting a pro-survival/tolerance mechanism against stress-related inflammation. Instead, in THP-1-derived macrophages, αS time-dependently potentiated the intracellular accumulation, and release of pro-inflammatory mediators. This was accompanied by mild toxicity, reduced autophagy-lysosomal markers, defective LDs formation, as well as impaired phagocytosis, and the appearance of stagnant lysosomes engulfed with phagocytosed cargo, suggesting a status of macrophage exhaustion reminiscent of hypophagia. Discussion: In summary, despite an apparently similar pro-inflammatory phenotype, monocytes and macrophages respond differently to intracellular αS accumulation in terms of cell survival, metabolism, and functions. Our results suggest that in periphery, αS exerts cell- and context-specific biological effects bridging alterations of autophagy, lipid dynamics, and inflammatory pathways.

Monocyte-Derived Chicken Macrophages Exposed to Eimeria tenella Sporozoites Display Reduced Susceptibility to Invasion by Toxoplasma gondii Tachyzoite.

Both Eimeria tenella and Toxoplasma gondii are common apicomplexan parasites in chickens. Host cell invasion by both protozoans includes gliding motility, host cell attachment and active penetration. Chicken macrophages as phagocytic cells participate in the innate host immune response against these two parasites. In this study, primary chicken monocyte-derived macrophages (MM) were infected with both pathogens to investigate mutual and host-parasite interactions. MM cultures were assigned to groups that were infected with E. tenella, T. gondii or both. In co-infected cultures, MM were first exposed to E. tenella sporozoites for 2 h. Afterwards, T. gondii tachyzoite infection was performed. Live-cell imaging was carried out to observe cell invasion and survival of T. gondii by single parasite tracking over a period of 20 h post infection (hpi). Quantitative analysis for parasite replication was performed by real-time quantitative PCR (qPCR) at 2, 6, 12 and 24 hpi. Overall, the ability of T. gondii to penetrate the cell membrane of the potential host cell was reduced, although high motility was displayed. We found that T. gondii tachyzoites adhered for more than 4 h to macrophages during early co-infection. qPCR results confirmed that significantly less T. gondii entered in E. tenella-activated MM at 2 hpi, and a reduced proportion of intracellular T. gondii survived and replicated in these cells at 24 hpi. We conclude that E. tenella modulates host cell responses to another apicomplexan agent, T. gondii, reducing active invasion and multiplication in chicken primary macrophages.

Resveratrol and its analogs suppress HIV replication, oxidative stress, and inflammation in macrophages.

HIV suppression in brain viral reservoirs, especially macrophages, and microglia is critical to suppress HIV neuropathogenesis and subsequently HIV-associated neurocognitive disorders (HAND). Since most antiretroviral therapy (ART) drugs do not achieve optimal therapeutic concentrations in the brain and can cause neurotoxicity, an alternative/adjuvant therapy is needed to suppress HIV neuropathogenesis. In this study, our objectives were to examine the anti-HIV, antioxidant, and anti-inflammatory potential of resveratrol (RES) and its synthetic analogs 4-(E)-{(p-tolylimino)-methylbenzene-1,2-diol} (TIMBD) and 4-(E)-{(4-hydroxyphenylimino)-methylbenzene,1,2-diol} (HPIMBD) in HIV-infected macrophages. We used HIV replication (viral load), oxidative stress (reactive oxygen species and antioxidant enzymes), and inflammatory response (pro- and anti-inflammatory cytokines/chemokines) assays to achieve the objectives of the study. Our results showed that RES and its analogs HPIMBD and TIMBD at 25 µM concentration significantly decrease HIV replication in both primary monocyte-derived macrophages and U1-differentiated macrophages. Moreover, RES and its analogs do not induce any cytotoxicity for up to 3 days in these cells. Further, treatment with RES and TIMBD (25 µM) also reduced the levels of reactive oxygen species without affecting the expression of antioxidant enzymes, SOD1, and catalase in U1 macrophages. Besides, RES and HPIMBD treatment inhibited the proinflammatory cytokines and chemokines in U1 macrophages, which was associated with decreased levels of anti-inflammatory cytokines. Importantly, our western blot experiments show that RES also decreases cellular proinflammatory cytokine IL-1β, which is usually elevated in both myeloid and neuronal cells upon HIV infection. Taken together, our results suggest that RES and/or its analogs are important adjuvants that may be used not only to suppress HIV but also oxidative stress and inflammation in brain viral reservoirs.

Vpx requires active cellular dNTP biosynthesis to effectively counteract the anti-lentivirus activity of SAMHD1 in macrophages

HIV-1 replication in primary monocyte-derived macrophages (MDMs) is kinetically restricted at the reverse transcription step due to the low deoxynucleoside triphosphates (dNTP) pools established by host dNTPase, SAM and HD domain containing protein 1 (SAMHD1). Lentiviruses such as HIV-2 and some Simian immunodeficiency virus counteract this restriction using viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates intracellular dNTP pools. However, how dNTP pools increase after Vpx degrades SAMHD1 in nondividing MDMs where no active dNTP biosynthesis is expected to exists remains unclear. In this study, we monitored known dNTP biosynthesis machinery during primary human monocyte differentiation to MDMs and unexpectedly found MDMs actively express dNTP biosynthesis enzymes such as ribonucleotide reductase, thymidine kinase 1, and nucleoside-diphosphate kinase. During differentiation from monocytes the expression levels of several biosynthesis enzymes are upregulated, while there is an increase in inactivating SAMHD1 phosphorylation. Correspondingly, we observed significantly lower levels of dNTPs in monocytes compared to MDMs. Without dNTP biosynthesis availability, Vpx failed to elevate dNTPs in monocytes, despite SAMHD1 degradation. These extremely low monocyte dNTP concentrations, which cannot be elevated by Vpx, impaired HIV-1 reverse transcription in a biochemical simulation. Furthermore, Vpx failed to rescue the transduction efficiency of a HIV-1 GFP vector in monocytes. Collectively, these data suggest that MDMs harbor active dNTP biosynthesis and Vpx requires this dNTP biosynthesis to elevate dNTP levels to effectively counteract SAMHD1 and relieve the kinetic block to HIV-1 reverse transcription in MDMs.

Transcriptional changes associated with apoptosis and type I IFN underlie the early interaction between Besnoitia besnoiti tachyzoites and monocyte-derived macrophages

Besnoitia besnoiti-infected bulls may develop severe systemic clinical signs and orchitis that may ultimately cause sterility during the acute infection. Macrophages might play a relevant role in pathogenesis of the disease and the immune response raised against B. besnoiti infection. This study aimed to dissect the early interaction between B. besnoiti tachyzoites and primary bovine monocyte-derived macrophages in vitro. First, the B. besnoiti tachyzoite lytic cycle was characterized. Next, dual transcriptomic profiling of B. besnoiti tachyzoites and macrophages was conducted at early infection (4 and 8 h p.i.) by high-throughput RNA sequencing. Macrophages inoculated with heat-killed tachyzoites (MO-hkBb) and non-infected macrophages (MO) were used as controls. Besnoitia besnoiti was able to invade and proliferate in macrophages. Upon infection, macrophage activation was demonstrated by morphological and transcriptomic changes. Infected macrophages were smaller, round and lacked filopodial structures, which might be associated with a migratory phenotype demonstrated in other apicomplexan parasites. The number of differentially expressed genes (DEGs) increased substantially during infection. In B. besnoiti-infected macrophages (MO-Bb), apoptosis and mitogen-activated protein kinase (MAPK) pathways were regulated at 4 h p.i., and apoptosis was confirmed by TUNEL assay. The Herpes simplex virus 1 infection pathway was the only significantly enriched pathway in MO-Bb at 8 h p.i. Relevant DEGs of the Herpes simplex virus 1 infection (IFNα) and the apoptosis pathways (CHOP-2) were also significantly regulated in the testicular parenchyma of naturally infected bulls. Furthermore, the parasite transcriptomic analysis revealed DEGs mainly related to host cell invasion and metabolism. These results provide a deep overview of the earliest macrophage modulation by B. besnoiti that may favour parasite survival and proliferation in a specialized phagocytic immune cell. Putative parasite effectors were also identified.

Optimization of CRISPR-Cas9 Mediated Knockout Protocol in Primary Human Myeloid Cells

Abstract Myeloid cells are an important player in the immune system, and form the first line of defense against pathogens, such as Mycobacterium tuberculosis (Mtb), the causative pathogen for tuberculosis (TB) disease. They include macrophages, a target of Mtbinfection, and dendritic cells (DCs), which present Mtb-derived antigens to prime and activate T cells. In order to investigate myeloid-specific host factors involved in the response to Mtbinfection, we sought to optimize a protocol to genetically edit primary human monocyte-derived DCs and macrophages for subsequent infection experiments with Mtb. We built on prior published protocol by Hiatt, et al. to edit monocyte-derived DCs and macrophages using nucleofection with ribonucleoproteins (RNP), which are Cas9 recombinant protein complexed with multiplexed guide RNA (gRNA) molecules. We tested reaction volume, RNP concentration, rest time, and nucleofection programs. We found that a molar ratio of Cas9:gRNA was most efficient at 1:2 at 1.8μM final RNP concentration in combination with a 2hr rest time after nucleofection before adding differentiation supplement. Lonza nucleofection program EA100 proved to be the optimal program with the most recovered cells. In conclusion, we optimized a robust protocol for editing and culturing edited primary monocyte-derived DCs and macrophages. This opens opportunities for interrogation of candidate TB risk genes such as Fumarylacetoacetate hydrolase (FAH). These cells can now be used for mechanistic studies of Mtbinfection to define mechanisms of pathogenesis.

Development of an Inflammation-Triggered In Vitro "Leaky Gut" Model Using Caco-2/HT29-MTX-E12 Combined with Macrophage-like THP-1 Cells or Primary Human-Derived Macrophages.

The "leaky gut" syndrome describes a damaged (leaky) intestinal mucosa and is considered a serious contributor to numerous chronic diseases. Chronic inflammatory bowel diseases (IBD) are particularly associated with the "leaky gut" syndrome, but also allergies, autoimmune diseases or neurological disorders. We developed a complex in vitro inflammation-triggered triple-culture model using 21-day-differentiated human intestinal Caco-2 epithelial cells and HT29-MTX-E12 mucus-producing goblet cells (90:10 ratio) in close contact with differentiated human macrophage-like THP-1 cells or primary monocyte-derived macrophages from human peripheral blood. Upon an inflammatory stimulus, the characteristics of a "leaky gut" became evident: a significant loss of intestinal cell integrity in terms of decreased transepithelial/transendothelial electrical resistance (TEER), as well as a loss of tight junction proteins. The cell permeability for FITC-dextran 4 kDa was then increased, and key pro-inflammatory cytokines, including TNF-alpha and IL-6, were substantially released. Whereas in the M1 macrophage-like THP-1 co-culture model, we could not detect the release of IL-23, which plays a crucial regulatory role in IBD, this cytokine was clearly detected when using primary human M1 macrophages instead. In conclusion, we provide an advanced human in vitro model that could be useful for screening and evaluating therapeutic drugs for IBD treatment, including potential IL-23 inhibitors.

Abstract 3471: A function-based high-throughput discovery platform, myeloid iOTarg, identifies novel immune checkpoints of the tumor microenvironment

Abstract Tumor-associated macrophages (TAMs) are important players to maintain immunosuppression within the tumor microenvironment (TME) and attenuate the function of effector immune cells to promote tumor survival. High density of TAMs is well-recognized as a feature of tumor progression and poor prognostic factor across various tumor types. Therefore, therapeutic strategies to dampen TAMs’ suppressive potential in TME and reprogram them towards a pro-inflammatory phenotype are being increasingly appreciated as being paramount for an effective immunotherapy. Nevertheless, the key mediators of TAM-induced immunosuppression remain largely unknown, identification of which can lead to the development of novel immunotherapeutic strategies aimed at re-educating the immunosuppressive TAMs to an anti-tumor phenotype. To systematically interrogate the genes responsible for the immune-suppressive phenotype of TAMs, we developed a functional, high-throughput genetic screening platform (iOTarg™) based on human primary monocyte-derived M2-like macrophages. Using CRISPR, we knocked out a proprietary library of well-expressed and druggable genes (1400 genes) in two independent donor-derived M2-like macrophages and cocultured them with activated autologous effector T cells (TCs). We achieved nearly complete knockout (KO) efficacy over a range of control genes. Functional impact of individual KO on macrophage viability and phenotype, as well as on TC activity, was measured using multiparametric assay readouts. Initial hits were reconfirmed in three additional donors in a secondary screen that integrated an additional tumor lysis readout. As expected, knockout of CSF1R, which is essential for macrophage maintenance, resulted in a dramatic loss of macrophage cell viability, whereas KO of established TAM markers, TREM2 and Clever-1, induced a change in macrophage phenotype. Furthermore, KO of immune-inhibitory receptor LILRB2 reduced the M2-like phenotype and restored TC activity. Inhibition of M2 activation of macrophages and subsequent increase in TC activation culminated in strong tumor cell killing for a subset of genes, highlighting an untapped repertoire of novel TAM-associated immune-checkpoint targets. Taken together, we report for the first time a highly sophisticated target discovery platform that addresses the functional role of any TAM-expressed gene in regulating macrophage viability, phenotype, T cell activity and even its gross impact on tumor cell lysis, all done in a high-throughput format employing CRISPR-edited primary human immune cells and multi-parametric functional immunological assays. As a result, we could confirm well-known targets in clinical testing, as well as identify additional novel targets that could lead to first-in-class, TME-based therapeutics and expansion of treatment options in immune oncology. Citation Format: Kritika Sudan, Tillmann Michels, Carmen Amerhauser, Claudia Tschulik, Leonie Majunke, Lucille Albert, Valentina Volpin, Adriana Turqueti-Neves, Ronny Milde, Nisit Khandelwal. A function-based high-throughput discovery platform, myeloid iOTarg, identifies novel immune checkpoints of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3471.

Using microfluidic models to evaluate whether monocyte preconditioning plays a role in tumor-associated macrophage mediated resistance to hormone therapy in men with prostate cancer.

228 Background: While men with metastatic castrate-resistant prostate cancer benefit greatly from 2nd generation androgen targeted therapies (ATTs), the utility of these therapies is inevitably limited by the development of resistance. Tumor associated macrophages (TAMs) are a myeloid immune cell population within the prostate tumor microenvironment (TME) that have been shown to play an integral role in development of ATT-resistance. However, the pathways that underlie TAM-mediated ATT resistance remain under investigation. In particular, many TAMs within the TME are derived from circulating monocytes and it is not yet clear whether the pathways driving TAM-mediated resistance are activated exclusively within the TME or if the activation of critical resistance pathways begins while the cells are circulating as monocytes. In this study, we utilized novel microscale technologies to investigate the transcriptome of monocytes and macrophages derived from patients progressing and responding to ATTs as well as the degree to which the macrophages promote ATT resistance in prostate tumor models. Methods: Primary monocytes were isolated from 22 men with prostate cancer who were either progressing or responding to 2ndgeneration ATTs. A portion of the monocytes from each patient were isolated for transcriptome analysis and the rest were differentiated into monocyte-derived macrophages (MDMs). MDMs were then cultured in monoculture or in co-culture with androgen-independent prostate tumor cells (22Rv1 and C42B). Each culture was then left untreated or treated with enzalutamide therapy. Following culture/treatment, each macrophage condition was isolated for transcriptome analysis and tumor cell populations from each condition were evaluated for treatment cytotoxicity. Results: Viability data from MDM-C42B co-culture demonstrated a 20% increase in tumor cell viability following enzalutamide treatment when C42B cells were cultured with primary MDMs from castrate-sensitive patients as compared to MDMs from castrate-resistant patients. Subsequent transcriptome analysis demonstrated key signatures in monocyte/macrophage populations that were associated ATT response. Conclusions: Leveraging a novel microfluidic culture platform known as Stacks, we were able to utilize primary, patient-derived cells to demonstrate that the role of TAMs in ATT resistance within the prostate TME is dependent, at least in part, on factors that are present prior to extravasation into the TME. Transcriptome analysis identified candidate pathways, which may be driving TAM-mediated differences in ATT response. These findings have the potential to lead to future use of circulating monocytes as a predictive biomarker as well as lead to the identification and targeting of ATT resistance pathways in patients.

Preparation of human primary macrophages to study the polarization from monocyte-derived macrophages to pro- or anti-inflammatory macrophages at biomaterial interface in vitro

Testing of dental materials when in contact with innate immune cells has been so far hindered by the lack of proper invitro models. Human primary monocyte-derived macrophages (MDMs) would be an excellent option to this aim. However, the inability to detach them from the tissue culture plates contrast the possibility to culture them on biomaterials. The goal of the present work is to present and validate an innovative protocol to obtain MDMs from peripheral blood monocytes, and to reseed them in contact with biomaterials without altering their viability and phenotype. We differentiated MDMs on ultra-low attachment tissue culture plastics and recovered them with specific detachment solution in order to be reseeded on a secondary substrate. Therefore, using biological assays (RT-PCR, Western blot, and immunofluorescence) we compared their phenotype to MDMs differentiated on standard culture plates. Transferred MDMs keep their differentiated M0 resting state, as well as the ability to be polarized into M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages. These data provide the dental material research community the unprecedented possibility to investigate the immunomodulatory properties of biomaterials for dental application.

Impact of latency-reversing agents on human macrophage physiology.

HIV-1 eradication is hindered by the presence of inducible long-lived reservoirs of latently infected cells which rapidly disseminate viral particles upon treatment interruption. Eliminating these reservoirs by the so-called shock and kill strategy represents a crucial concept toward an HIV-1 cure. Several molecules called latency-reversing agents (LRAs) are under intensive investigations to reactivate virus gene expression. These studies are mainly conducted on CD4+ T cells where LRAs are well tolerated and did not induce global cellular activation. However, despite their broad spectrum, the putative impact of LRAs on other cellular reservoirs such as macrophages is still ill-defined. We investigated the impact of the protein kinase C (PKC) activator bryostatin-1, bromodomain inhibitor JQ1 and histone deacetylase inhibitor romidepsin used either alone or in combination on human primary monocyte-derived macrophages (MDMs). We demonstrate that bryostatin-1, JQ1, and romidepsin or their combinations are not toxic at nanomolar concentrations but induce metabolic and morphologic alterations of MDMs. Bryostatin-1 triggered the secretion of pro-inflammatory cytokines, while JQ-1 decreased it. Phagocytosis and endocytosis were modestly impaired upon bryostatin-1 treatment whereas efferocytosis was markedly downregulated by romidepsin. Despite its pro-inflammatory profile, bryostatin-1 did not induce classically activated macrophage markers. Finally, we reveal that conditioned medium from bryostatin-1-treated macrophages did not potentiate its reactivation feature. Our study reveals that LRAs can diversely impact basic physiologic features of human primary macrophages and could potentially decrease reactivation of nearby CD4+ T cells latently infected with HIV-1. Our observations further stress the need to include different cell populations when assessing HIV-1 cure strategies.

SRSF1 acts as an IFN-I-regulated cellular dependency factor decisively affecting HIV-1 post-integration steps.

Efficient HIV-1 replication depends on balanced levels of host cell components including cellular splicing factors as the family of serine/arginine-rich splicing factors (SRSF, 1-10). Type I interferons (IFN-I) play a crucial role in the innate immunity against HIV-1 by inducing the expression of IFN-stimulated genes (ISGs) including potent host restriction factors. The less well known IFN-repressed genes (IRepGs) might additionally affect viral replication by downregulating host dependency factors that are essential for the viral life cycle; however, so far, the knowledge about IRepGs involved in HIV-1 infection is very limited. In this work, we could demonstrate that HIV-1 infection and the associated ISG induction correlated with low SRSF1 levels in intestinal lamina propria mononuclear cells (LPMCs) and peripheral blood mononuclear cells (PBMCs) during acute and chronic HIV-1 infection. In HIV-1-susceptible cell lines as well as primary monocyte-derived macrophages (MDMs), expression levels of SRSF1 were transiently repressed upon treatment with specific IFNα subtypes in vitro. Mechanically, 4sU labeling of newly transcribed mRNAs revealed that IFN-mediated SRSF1 repression is regulated on early RNA level. SRSF1 knockdown led to an increase in total viral RNA levels, but the relative proportion of the HIV-1 viral infectivity factor (Vif) coding transcripts, which is essential to counteract APOBEC3G-mediated host restriction, was significantly reduced. In the presence of high APOBEC3G levels, however, increased LTR activity upon SRSF1 knockdown facilitated the overall replication, despite decreased vif mRNA levels. In contrast, SRSF1 overexpression significantly impaired HIV-1 post-integration steps including LTR transcription, alternative splice site usage, and virus particle production. Since balanced SRSF1 levels are crucial for efficient viral replication, our data highlight the so far undescribed role of SRSF1 acting as an IFN-modulated cellular dependency factor decisively regulating HIV-1 post-integration steps.