Recruitment Of Innate Immune Cells Research Articles

Exploring Microbial Metabolite Receptors in Inflammatory Bowel Disease: An In Silico Analysis of Their Potential Role in Inflammation and Fibrosis.

Metabolites produced by dysbiotic intestinal microbiota can influence disease pathophysiology by participating in ligand-receptor interactions. Our aim was to investigate the differential expression of metabolite receptor (MR) genes between inflammatory bowel disease (IBD), healthy individuals (HIs), and disease controls in order to identify possible interactions with inflammatory and fibrotic pathways in the intestine. RNA-sequencing datasets containing 643 Crohn's disease (CD) patients, 467 ulcerative colitis (UC) patients and 295 HIs, and 4 Campylobacter jejuni-infected individuals were retrieved from the Sequence Read Archive, and differential expression was performed using the RaNA-seq online platform. The identified differentially expressed MR genes were used for correlation analysis with up- and downregulated genes in IBD, as well as functional enrichment analysis using a R based pipeline. Overall, 15 MR genes exhibited dysregulated expression in IBD. In inflamed CD, the hydroxycarboxylic acid receptors 2 and 3 (HCAR2, HCAR3) were upregulated and were associated with the recruitment of innate immune cells, while, in the non-inflamed CD ileum, the cannabinoid receptor 1 (CNR1) and the sphingosine-1-phospate receptor 4 (S1PR4) were downregulated and were involved in the regulation of B-cell activation. In inflamed UC, the upregulated receptors HCAR2 and HCAR3 were more closely associated with the process of TH-17 cell differentiation, while the pregnane X receptor (NR1I2) and the transient receptor potential vanilloid 1 (TRPV1) were downregulated and were involved in epithelial barrier maintenance. Our results elucidate the landscape of metabolite receptor expression in IBD, highlighting associations with disease-related functions that could guide the development of new targeted therapies.

Combination of 5-fluorouracil with photodynamic therapy enhances immune responses in a murine model of actinic keratosis

SignificancePhotodynamic therapy (PDT) to treat actinic keratoses (AK) involves application of a photosensitizer followed by exposure to light, resulting in effective lesion clearance by inducing cell death and long-term immune responses. Here, we show that pretreatment of AK lesions with 5-fluorouracil (5FU) causes significant enhancement of PDT-related immune effects. ApproachSKH-1 mice with AK (generated by repeated UVB exposure) were treated topically with 5FU for three days prior to application of topical ALA followed by blue light exposure. AK lesions were harvested for time-course analyses of intra-tumoral immune responses after PDT with or without 5FU. ResultsImmunofluorescence data showed that 5FU pretreatment increased recruitment of innate immune cells, infiltration of activated T cells and of cytotoxic T cells and reduced the presence of cells expressing the immune checkpoint marker PD1. ConclusionsOur results suggest that a combination of 5FU and PDT may synergize to provide better management of AK in the dermatology clinic.

Abstract 5248: Novel costimulatory domain improves CAR-monocyte activity against tumors

Abstract Myeloid cells, unlike other immune cells such as T cells or NK cells, are known residents in the solid tumor microenvironment (TME). In the absence of checkpoints and in proinflammatory conditions, myeloid cells (particularly M1 macrophages) are known to be capable of direct phagocytosis of tumor cells and can present tumor-associated antigens to the host immune system. However, the immunosuppressive conditions within the TME restrict and limit the anti-tumor response of tumor associated macrophages (TAMs), including their ability to recruit and activate other immune cells against the tumor. Engineered CAR-Monocytes can serve a unique function in cell therapy by bridging a key gap in the treatment of solid tumors. We are developing an autologous engineered CAR-M cell therapy product targeting Glypican-3 (GPC3) to treat hepatocellular carcinoma (HCC). The CAR serves a dual purpose as a homing ‘GPS’ signal for trafficking directly to the tumor site, and for directing phagocytosis specifically at targeted tumor cells. We have designed a proprietary CAR molecule (M83 CAR) which contains a macrophage-specific costimulatory domain that significantly increases the phagocytosis function of the CAR-M cells. Furthermore, we have demonstrated that the M83 CAR-M cells are not inhibited by the prevalent CD47 checkpoint (‘do not eat me’ signal expressed on tumor cells), which is known to restrict myeloid function in the TME. Effective phagocytosis of engineered CAR-M cells is central to subsequent mechanisms of actions that can elicit robust anti-tumor immunity against patient-specific neoantigens. However, the first barrier to overcome is effective infiltration of engineered CAR-M into the tumor from the periphery. We have demonstrated that our engineered CAR-Monocyte drug product can successfully home to the targeted tumor specifically, from the periphery. Subsequent in situ differentiation of CAR-monocytes into CAR-macrophages (M1) enables robust tumor cell phagocytosis, the central mechanism that can result in the following series of events: 1) proinflammatory shift in the TME milieu, 2) recruitment of innate and adaptive immune cells, 3) activation of T-cells against tumor neo-antigens. This is a unique outcome of the CAR-M mechanism of action that is not capitulated by CAR-T or CAR-NK cells. Leveraging and further enhancing CAR-M function could lead to the rejection of the tumor and its metastases, particularly in combination with other immune-modulating therapies. Citation Format: Kevin Carbajal, Warren Anderson, Kilsoo Jeon, Grace Reynolds, Prasanna Ekambaram, Beatrice Breckheimer, Anastasya Birger, Jessica Pafford, Matthew Esquivel, Adam Pecoraro, Sumiti Jain. Novel costimulatory domain improves CAR-monocyte activity against tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5248.

Macrophages coordinate immune response to laser-induced injury via extracellular traps.

Retinal degeneration results from disruptions in retinal homeostasis due to injury, disease, or aging and triggers peripheral leukocyte infiltration. Effective immune responses rely on coordinated actions of resident microglia and recruited macrophages, critical for tissue remodeling and repair. However, these phagocytes also contribute to chronic inflammation in degenerated retinas, yet the precise coordination of immune response to retinal damage remains elusive. Recent investigations have demonstrated that phagocytic cells can produce extracellular traps (ETs), which are a source of self-antigens that alter the immune response, which can potentially lead to tissue injury. Innovations in experimental systems facilitate real-time exploration of immune cell interactions and dynamic responses. We integrated in vivo imaging with ultrastructural analysis, transcriptomics, pharmacological treatments, and knockout mice to elucidate the role of phagocytes and their modulation of the local inflammatory response through extracellular traps (ETs). Deciphering these mechanisms is essential for developing novel and enhanced immunotherapeutic approaches that can redirect a specific maladaptive immune response towards favorable wound healing in the retina. Our findings underscore the pivotal role of innate immune cells, especially macrophages/monocytes, in regulating retinal repair and inflammation. The absence of neutrophil and macrophage infiltration aids parenchymal integrity restoration, while their depletion, particularly macrophages/monocytes, impedes vascular recovery. We demonstrate that macrophages/monocytes, when recruited in the retina, release chromatin and granular proteins, forming ETs. Furthermore, the pharmacological inhibition of ETosis support retinal and vascular repair, surpassing the effects of blocking innate immune cell recruitment. Simultaneously, the absence of ETosis reshapes the inflammatory response, causing neutrophils, helper, and cytotoxic T-cells to be restricted primarily in the superficial capillary plexus instead of reaching the damaged photoreceptor layer. Our data offer novel insights into innate immunity's role in responding to retinal damage and potentially help developing innovative immunotherapeutic approaches that can shift the immune response from maladaptive to beneficial for retinal regeneration.

Advances in lung ischemia/reperfusion injury: unraveling the role of innate immunity.

Lung ischemia/reperfusion injury (LIRI) is a common occurrence in clinical practice and represents a significant complication following pulmonary transplantation and various diseases. At the core of pulmonary ischemia/reperfusion injury lies sterile inflammation, where the innate immune response plays a pivotal role. This review aims to investigate recent advancements in comprehending the role of innate immunity in LIRI. A computer-based online search was performed using the PubMed database and Web of Science database for published articles concerning lung ischemia/reperfusion injury, cell death, damage-associated molecular pattern molecules (DAMPs), innate immune cells, innate immunity, inflammation. During the process of lung ischemia/reperfusion, cellular injury even death can occur. When cells are injured or undergo cell death, endogenous ligands known as DAMPs are released. These molecules can be recognized and bound by pattern recognition receptors (PRRs), leading to the recruitment and activation of innate immune cells. Subsequently, a cascade of inflammatory responses is triggered, ultimately exacerbating pulmonary injury. These steps are complex and interrelated rather than being in a linear relationship. In recent years, significant progress has been made in understanding the immunological mechanisms of LIRI, involving novel types of cell death, the ability of receptors other than PRRs to recognize DAMPs, and a more detailed mechanism of action of innate immune cells in ischemia/reperfusion injury (IRI), laying the groundwork for the development of novel diagnostic and therapeutic approaches. Various immune components of the innate immune system play critical roles in lung injury after ischemia/reperfusion. Preventing cell death and the release of DAMPs, interrupting DAMPs receptor interactions, disrupting intracellular inflammatory signaling pathways, and minimizing immune cell recruitment are essential for lung protection in LIRI.

Bile acids induce IL-1α and drive NLRP3 inflammasome-independent production of IL-1β in murine dendritic cells.

Bile acids are amphipathic molecules that are synthesized from cholesterol in the liver and facilitate intestinal absorption of lipids and nutrients. They are released into the small intestine upon ingestion of a meal where intestinal bacteria can modify primary into secondary bile acids. Bile acids are cytotoxic at high concentrations and have been associated with inflammatory diseases such as liver inflammation and Barrett's Oesophagus. Although bile acids induce pro-inflammatory signalling, their role in inducing innate immune cytokines and inflammation has not been fully explored to date. Here we demonstrate that the bile acids, deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) induce IL-1α and IL-1β secretion in vitro in primed bone marrow derived dendritic cells (BMDCs). The secretion of IL-1β was found not to require expression of NLRP3, ASC or caspase-1 activity; we can't rule out all inflammasomes. Furthermore, DCA and CDCA were shown to induce the recruitment of neutrophils and monocytes to the site of injection an intraperitoneal model of inflammation. This study further underlines a mechanistic role for bile acids in the pathogenesis of inflammatory diseases through stimulating the production of pro-inflammatory cytokines and recruitment of innate immune cells.

TMIC-59. IL-2 SECRETING ENGINEERED MYELOID CELLS POTENTIATE CYTOTOXIC EFFECTOR CELL ACTIVATION TO DELAY MALIGNANT PROGRESSION IN A PRECLINICAL MODEL OF GLIOMA

Abstract INTRODUCTION Gliomas are the most prevalent brain tumor in the adolescent and young adult population (AYA) and one of the leading causes of cancer-related death. In fact, two-thirds of glioma AYA patients are affected by low-grade gliomas (LGGs). Moreover, there are no therapies to prevent glioma LGG malignant progression. Malignant progression is associated with a significant reduction of tumor infiltrating cytotoxic effector immune cells, primarily due to the immunosuppressive tumor microenvironment (TME) mediated by regulatory T lymphocytes, tumor associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs). To that end, immunotherapies have had limited efficacy in improving outcomes for primary and secondary HGGs. HYPOTHESIS We hypothesize that engineered myeloid cells that release IL-2 will enhance the trafficking of activated effector T cells and potentiate a pro-inflammatory cascade in the low-grade glioma TME, delaying malignant progression. METHODS Immunocompetent RCAS n/tv-a LGG bearing animals were retro-orbitally treated at day 21 with a single dose of bone-marrow derived myeloid cells, engineered for the release of Interlekin-2 (GEMys-IL2). The inflammatory status of the TME along with the composition and functional status of tumor infiltrating immune cells were investigated in vivo by cytokine array, mass cytometry, and bulk RNA sequencing. RESULTS Our complementary dataset demonstrated that GEMys-IL2 infiltrated the glioma microenvironment and triggered a pro-inflammatory cascade involving the upregulation and release of IL2, IFNy, CXCL9, Tim1, CD26 and other pro-inflammatory cytokines associated with the activation and recruitment of innate and adaptive immune cells within the TME. In addition, we observed a downregulation of immunosuppressive signaling pathways such as IL10, PPAR, LXR/RXR, CTLA4 and PD-1. Also, a single dose of GEMys-IL2 systemically injected in RCAS/t-va immunocompetent LGG bearing mice potentiated pro-inflammatory remodeling of the TME and significantly improved overall survival. CONCLUSIONS These preclinical studies highlight a novel approach in developing next-generation innate immunotherapies.

Mechanisms and therapeutic strategies to combat the recurrence and progression of hepatocellular carcinoma after thermal ablation

Thermal ablation (TA), including radiofrequency ablation (RFA) and microwave ablation (MWA), has become the main treatment for early-stage hepatocellular carcinoma (HCC) due to advantages such as safety and minimal invasiveness. However, HCC is prone to local recurrence, with more aggressive malignancies after TA closely related to TA-induced changes in epithelial-mesenchymal transition (EMT) and remodeling of the tumor microenvironment (TME). According to many studies, various components of the TME undergo complex changes after TA, such as the recruitment of innate and adaptive immune cells, the release of tumor-associated antigens (TAAs) and various cytokines, the formation of a hypoxic microenvironment, and tumor angiogenesis. Changes in the TME after TA can partly enhance the anti-tumor immune response; however, this response is weak to kill the tumor completely. Certain components of the TME can induce an immunosuppressive microenvironment through complex interactions, leading to tumor recurrence and progression. How the TME is remodeled after TA and the mechanism by which the TME promotes HCC recurrence and progression are unclear. Thus, in this review, we focused on these issues to highlight potentially effective strategies for reducing and preventing the recurrence and progression of HCC after TA.

Immunostimulatory effects of marine algae extracts on in vitro antigen-presenting cell activation and in vivo immune cell recruitment.

Marine algae are photosynthetic eukaryotic organisms that are widely used as sources of food, cosmetics, and drugs. However, their biological and immunological effects on immune cells have not been fully elucidated. To unravel their immunological activity and broaden their application, we generated antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages, from mouse bone marrow cells and treated them with six different marine algae extracts (MAEs). We evaluated cell viability, activation marker expression, and pro-inflammatory cytokine production by APCs after 2 days of MAE treatment. All six MAEs significantly induced cytokine production of APCs, among which Pyropia yezoensis (PY), Peyssonnelia caulifera (PC), and Meristotheca papulosa (MP) extracts exhibited the strongest effect. Cladophora wrightiana var. minor (CW) extract moderately upregulated cytokine levels but increased the expression of activation markers on DCs. Moreover, PY, PC, MP, Sargassum pectinifera (SP), and Caulerpa okamurae (CO) pre-treated APCs effectively stimulated T-cell proliferation and cytokine production. Furthermore, the mice injected with MAEs exhibited higher cytokine (TNF-α, IL-6, and IL-1β) production as well as enhanced innate immune cell recruitment capacities (DCs, monocytes, neutrophils, and natural killer cells) in the peritoneal cavity of the mice compared to those of the non-treated mice. Therefore, all MAEs exhibited immunostimulatory potential, with PY, PC, CW, and MP extracts being the most effective in stimulating immune responses and cell activation. To the best of our knowledge, this is the first study to determine the immunomodulatory activities of six MAEs both in vitro and in vivo.

Infection with Influenzavirus A in a murine model induces epithelial bronchial lesions and distinct waves of innate immune-cell recruitment.

Inflammatory lesions after Influenza A viruses (IAV) are potential therapeutic target for which better understanding of post-infection immune mechanisms is required. Most studies to evaluate innate immune reactions induced by IAV are based on quantitative/functional methods and anatomical exploration is most often non-existent. We aimed to study pulmonary damage and macrophage recruitment using two-photon excitation microscopy (TPEM) after IAV infection. We infected C57BL/6 CD11c+YFP mice with A/Puerto Ricco/8/34 H1N1. We performed immune cell analysis, including flow cytometry, cytokine concentration assays, and TPEM observations after staining with anti-F4/80 antibody coupled to BV421. We adapted live lung slice (LLS) method for ex-vivo intravital microscopy to analyze cell motility. TPEM provided complementary data to flow cytometry and cytokine assays by allowing observation of bronchial epithelium lesions and spreading of local infection. Addition of F4/80-BV421 staining allowed us to precisely determine timing of recruitment and pulmonary migration of macrophages. Ex-vivo LLS preserved cellular viability, allowing us to observe acceleration of macrophage motility. After IAV infection, we were able to explore structural consequences and successive waves of innate immune cell recruitment. By combining microscopy, flow cytometry and chemokine measurements, we describe novel and precise scenario of innate immune response against IAV.

Fibroblasts - the cellular choreographers of wound healing.

Injuries to our skin trigger a cascade of spatially- and temporally-synchronized healing processes. During such endogenous wound repair, the role of fibroblasts is multifaceted, ranging from the activation and recruitment of innate immune cells through the synthesis and deposition of scar tissue to the conveyor belt-like transport of fascial connective tissue into wounds. A comprehensive understanding of fibroblast diversity and versatility in the healing machinery may help to decipher wound pathologies whilst laying the foundation for novel treatment modalities. In this review, we portray the diversity of fibroblasts and delineate their unique wound healing functions. In addition, we discuss future directions through a clinical-translational lens.

TRIM32 reduced the recruitment of innate immune cells and the killing capacity of Listeria monocytogenes by inhibiting secretion of chemokines

Listeria monocytogenes (Lm) is a facultative, intracellular Gram-positive pathogenic bacterium that causes sepsis, a condition characterized by persistent excessive inflammation and organ dysfunction. However, the pathogenesis of Lm-induced sepsis is unknown. In this research, we discovered that TRIM32 is required for innate immune regulation during Lm infection. Trim32 deficiency remarkably reduced bacteremia and proinflammatory cytokine secretion in mice with severe Lm infection, preventing sepsis. Trim32−/− mice had a lower bacterial burden after Lm infection and survived significantly longer than wild-type (WT) mice, as well as lower serum levels of inflammatory cytokines TNF-α, IL-6, IL-18, IL-12p70, IFN-β, and IFN-γ at 1 day post infection (dpi) compared to WT mice. On the other hand, the chemokines CXCL1, CCL2, CCL7, and CCL5 were enhanced at 3 dpi in Trim32−/− mice than WT mice, reflecting increased recruitment of neutrophils and macrophages. Furthermore, Trim32−/− mice had higher levels of macrophage-associated iNOS to kill Lm. Collectively, our findings suggest that TRIM32 reduces innate immune cells recruitment and Lm killing capabilities via iNOS production.Graphical

MTOR inhibition suppresses Myc-driven polyposis by inducing immunogenic cell death

Myc is a key driver of colorectal cancer initiation and progression, but remains a difficult drug target. In this study, we show that mTOR inhibition potently suppresses intestinal polyp formation, regresses established polyps, and prolongs lifespan of APCMin/+ mice. Everolimus in diet strongly reduces p-4EBP1, p-S6, and Myc levels, and induces apoptosis of cells with activated β-catenin (p-S552) in the polyps on day 3. The cell death is accompanied by ER stress, activation of the extrinsic apoptotic pathway, innate immune cell recruitment, and followed by T-cell infiltration on day 14 persisting for months thereafter. These effects are absent in normal intestinal crypts with physiologic levels of Myc and a high rate of proliferation. Using normal human colonic epithelial cells, EIF4E S209A knockin and BID knockout mice, we found that local inflammation and antitumor efficacy of Everolimus requires Myc-dependent induction of ER stress and apoptosis. These findings demonstrate mTOR and deregulated Myc as a selective vulnerability of mutant APC-driven intestinal tumorigenesis, whose inhibition disrupts metabolic and immune adaptation and reactivates immune surveillance necessary for long-term tumor control.

CD154:CD11b blockade increases MPEC differentiation of virus-specific CD8 +T cells

Abstract CD154 pathway blockade has been shown to significantly improve graft survival in transplantation. Recently, CD11b was identified as an alternate receptor for CD154. CD154:CD11b interactions promote rejection by increasing the recruitment of innate and adaptive immune cells into the allograft. However, the effects of CD154:CD11b blockade on the protective immune response to infection during immunosuppression has not been elucidated. To address this, MHV68, a murine homolog of EBV, was used to assess the antigen-specific CD8 +T cell response. Naïve B6 mice were infected with MHV68 and treated on days 0, 2, 4, and 6 with a peptide inhibitor of CD154:CD11b binding (cM7). cM7 treatment resulted in an increase in tetramer +antigen-specific CD8 +T cells 10 days post-infection that exhibited an increase in gene expression associated with activation and antigen processing. To determine if this increase was a result of enhanced proliferation, CTV-labeled OT-I T cells were stimulated in vitro in the presence or absence of CD154:CD11b blockade. Results indicated that CD154:CD11b blockade did not impact proliferation. Instead, cM7 treatment in vitro resulted in an increase in the expression of CD69, CD25, and CD44, and in vivo resulted in an increase in CD127 hiKLRG1 loMPECs. In conclusion, these data demonstrate that CD154:CD11b blockade enhances the quantity and quality of virus-specific CD8 +T cells during protective immunity. We speculate that this could be due to the inhibition of terminal differentiation of antigen-specific T cells resulting in enhanced differentiation of high quality memory precursors, or could be due to an altered balance of CD154:CD40 vs CD154:CD11b binding, resulting in an increase in CD40-driven costimulation. Supported by grants from NIH (R01 AI152516)

Polyomavirus ependyma infection induces region-specific and Stat1-dependent expression of chemokines in the CNS

Abstract Immunity of the central nervous system to pathogens is partly established by critical barrier sites. One barrier within the four ventricles, the blood-cerebrospinal fluid (CSF)-brain barrier, is comprised of the choroid plexus epithelial cells separating the vasculature from the CSF, and the CSF from the brain parenchyma by the ependymal cells lining the ventricles. Intracerebroventricular delivery of mouse polyomavirus (MuPyV) in wild-type mice leads to infection of ependymal cells. In Stat1 null mice, viral infection is increased, and ependyma effacement is associated with hydrocephalus. Unknown is whether innate immunity and immune cell recruitment via Stat1 signaling by the ependyma limits MuPyV infection of the parenchyma. Dcdc2α-Cre mice, which show recombination in the ependyma and choroid plexus, were crossed with Stat1 fl/flmice to yield ependyma-specific Stat1 knockout. MuPyV infection was greater in Dcdc2αCre+ x Stat1 fl/flmice than in wild-type (WT) controls at 4 days post infection (dpi), but by 7 dpi the Dcdc2α-Cre+ x Stat1 fl/flmice exhibited higher levels of MuPyV infection in the periventricular region. Prior studies using the MuPyV-infected Stat1 null mice showed increased monocyte and T cell recruitment. Several chemokines (Ccl2, Cxcl10, Cxcl11, and Cxcl16) were elevated at 7 dpi in Dcdc2αCre+ x Stat1 fl/flmice relative to WT mice, while Cxcl13 was reduced at 4 and 7 dpi. Of these, Ccl2, Cxcl10 and Cxcl11 showed greater levels near the ventricles in both wild-type and Dcdc2αCre+ x Stat1 fl/flmice. Our data highlight the ependyma as a critical barrier protecting the brain from MuPyV infection that is dependent on Stat1 signaling. This work was supported by grants from the NIH (R35NS127217-01, T32CA60395-25).

Abstract 4892: MMAE drives immunomodulatory changes in a preclinical xenograft model that are distinct from other clinical-stage ADC payloads

Abstract Monomethyl auristatin E (MMAE), the payload delivered by vedotin ADCs, exerts its cytotoxic effects via microtubule disruption and induction of ER stress, leading to apoptosis and tumor cell death. In addition, MMAE also has the ability to induce immunogenic cell death (ICD) and immune modulation in the tumor microenvironment (TME) (Liu, 2020; Gray, 2020). Other clinical-stage and approved ADCs incorporate payloads that also cause microtubule disruption (DM1, DM4), or that drive DNA damage through topoisomerase I inhibition (exatecan) as the primary mechanism of action (MOA). While ADCs with different linker payloads produce clinical benefit, their long-term impact on survival and how they pair with PD(L)1 agents may differ based on their ability to elicit immune modulation. In this study we have investigated how these payloads compare to MMAE in their ability to drive immune changes in the TME and describe how these changes could support anti-tumor immunity and improve outcomes in the clinic.Building upon an initial in vitro assessment in which we observed superior induction of ICD markers and underlying ER stress with an MMAE ADC, we utilized a B7H4-expressing MDA-MB-468 xenograft model of triple-negative breast cancer to further characterize the ICD properties of ADC payloads. Tumor-bearing mice were treated with a single dose of B7H4-targeted antibody conjugated to either MMAE, DM1, DM4 or exatecan. Tumors were harvested seven days after treatment and processed for immunohistochemistry (IHC) to assess changes in immune cell infiltration and analyzed by RNA-seq to determine alterations in gene expression. Payload-driven anti-tumor activity was observed with all ADC treatments, each leading to a similar reduction in tumor volume compared to vehicle or unconjugated antibody. IHC staining of tumor sections for F4/80, a mouse macrophage marker, revealed that of the microtubule-disrupting agents, only MMAE significantly increased F4/80+ macrophage infiltration in both the tumor nests and stroma. Increased F4/80+ macrophages in tumor nests and stroma was also observed as a result of B7H4-exatecan ADC treatment. RNA-seq analysis revealed a unique profile for the MMAE conjugate, with an increase in human transcripts encoding cytokine and type I interferon response genes. In summary, the anti-tumor activity of the B7H4-MMAE ADC resulted in recruitment of mouse innate immune cells to the xenograft tumors. Importantly, immune response genes related to ICD were increased following B7H4-MMAE treatment compared to DM1, DM4, and exatecan conjugates. Together, these data suggest that treatment with vedotin ADCs results in robust immunomodulatory changes in vivo that are distinct from other clinical ADC payloads. Citation Format: Michelle Ulrich, Kerry Klussman, John J. Gosink, Sean Allred, Kelly Hensley, Piper M. Treuting, Nikhil J. Parekh, Elizabeth E. Gray, Shyra J. Gardai. MMAE drives immunomodulatory changes in a preclinical xenograft model that are distinct from other clinical-stage ADC payloads. [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 4892.

Single cell analysis reveals satellite cell heterogeneity for proinflammatory chemokine expression

Background: The expression of proinflammatory signals at the site of muscle injury are essential for efficient tissue repair and their dysregulation can lead to inflammatory myopathies. Macrophages, neutrophils, and fibroadipogenic progenitor cells residing in the muscle are significant sources of proinflammatory cytokines and chemokines. However, the inducibility of the myogenic satellite cell population and their contribution to proinflammatory signaling is less understood. Methods: Mouse satellite cells were isolated and exposed to lipopolysaccharide (LPS) to mimic sterile skeletal muscle injury and changes in the expression of proinflammatory genes was examined by RT-qPCR and single cell RNA sequencing. Expression patterns were validated in skeletal muscle injured with cardiotoxin by RT-qPCR and immunofluorescence. Results: Satellite cells in culture were able to express Tnfa, Ccl2, and Il6, within 2h of treatment with LPS. Single cell RNA-Seq revealed seven cell clusters representing the continuum from activation to differentiation. LPS treatment led to a heterogeneous pattern of induction of C-C and C-X-C chemokines (e.g., Ccl2, Ccl5, and Cxcl0) and cytokines (e.g., Tgfb1, Bmp2, Il18, and Il33) associated with innate immune cell recruitment and satellite cell proliferation. One cell cluster was enriched for expression of the antiviral interferon pathway genes under control conditions and LPS treatment. Activation of this pathway in satellite cells was also detectable at the site of cardiotoxin induced muscle injury. Conclusion: These data demonstrate that satellite cells respond to inflammatory signals and secrete chemokines and cytokines. Further, we identified a previously unrecognized subset of satellite cells that may act as sensors for muscle infection or injury using the antiviral interferon pathway.

Distinct systemic immune networks define severe vs. mild COVID-19 in hematologic and solid cancer patients.

The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, has impacted health across all sectors of society. A cytokine-release syndrome, combined with an inefficient response of innate immune cells to directly combat the virus, characterizes the severe form of COVID-19. While immune factors involved in the development of severe COVID-19 in the general population are becoming clearer, identification of the immune mechanisms behind severe disease in oncologic patients remains uncertain. Here we evaluated the systemic immune response through the analysis of soluble blood immune factors and anti-SARS-CoV-2 antibodies within the early days of a positive SARS-CoV-2 diagnostic in oncologic patients. Individuals with hematologic malignancies that went on to die from COVID-19 displayed at diagnosis severe leukopenia, low antibody production against SARS-CoV-2 proteins, and elevated production of innate immune cell recruitment and activation factors. These patients also displayed correlation networks in which IL-2, IL-13, TNF-alpha, IFN-gamma, and FGF2 were the focal points. Hematologic cancer patients that showed highly networked and coordinated anti-SARS-CoV-2 antibody production, with central importance of IL-4, IL-5, IL-12A, IL-15, and IL-17A, presented only mild COVID-19. Conversely, solid tumor patients that had elevated levels of inflammatory cytokines IL-6, CXCL8, and lost the coordinate production of anti-virus antibodies developed severe COVID-19 and died. Patients that displayed positive correlation networks between anti-virus antibodies, and a regulatory axis involving IL-10 and inflammatory cytokines recovered from the disease. We also provided evidence that CXCL8 is a strong predictor of death for oncologic patients and could be an indicator of poor prognosis within days of the positive diagnostic of SARS-CoV-2 infection. Our findings defined distinct systemic immune profiles associated with COVID-19 clinical outcome of patients with cancer and COVID-19. These systemic immune networks shed light on potential immune mechanisms involved in disease outcome, as well as identify potential clinically useful biomarkers.

Roles of Host Phospholipase D during Aspergillus fumigatus Infection in Mice

Objective: Aspergillus fumigatus infection in the lungs is accompanied by the recruitment of innate immune cells, phagocytosis, and the release of inflammatory factors. Phospholipase D (PLD) is a key regulator of cell migration and phagocytosis, but the effect of PLD deficiency on antifungal infection in animals is unknown. This study aims to investigate the impact of PLD on the host immune response to A. fumigatus infection under either immunocompetent or immunosuppressed status. Methods: The invasive pulmonary aspergillosis mouse model was created using a modified protocol with immunosuppression by steroids. For collection of bronchoalveolar lavage fluid (BALF) from mice, the lungs were washed eight times with 0.5 ml of PBS. Total cell counts in BALF were determined using a Coulter Counter. The content of alveolar macrophages, neutrophils, and monocytes in BALF was examined by flow cytometry and analyzed by FlowJo V10 software. Multiplex immunoassays were used to determine the concentrations of inflammatory cytokines in BALF. Results: In immunocompetent mice, alveolar macrophages were the major cell population in BALF after A. fumigatus infection, and a number of neutrophils and monocytes were recruited in the alveoli. Loss of both pld1 and pld2 genes did not affect the content of alveolar macrophages, neutrophils, or monocytes in BALF. Under immunosuppression induced by hydrocortisone acetate, pld1-/-pld2-/- mice showed higher mortality after A. fumigatus infection and had a higher fungal burden and much lower number of prominent focal areas of dense inflammatory infiltrates in lung tissue than wild type mice. Moreover, interleukin (IL)-12p40 significantly decreased, and IL-10 markedly increased, in BALF from pld1 -/- pld2 -/- mice after infection. Conclusion: Our findings revealed that, during A. fumigatus infection, deficiency in both pld1 and pld2 in mice was not conducive to the infiltration of inflammatory cells into lung tissue but promoted the release of IL-10 and blocked the release of IL-12, thereby increasing fungal burden and mortality.

Ras-Related Protein Rab5a Regulates Complement C5a Receptor Trafficking, Chemotaxis, and Chemokine Secretion in Human Macrophages

Complement activation and Rab GTPase trafficking are commonly observed in inflammatory responses. Recruitment of innate immune cells to sites of infection or injury and secretion of inflammatory chemokines are promoted by complement component 5a (C5a) that activates the cell surface protein C5a receptor1 (C5aR1). Persistent activation can lead to a myriad of inflammatory and autoimmune diseases. Here, we demonstrate that the mechanism of C5a induced chemotaxis of human monocyte-derived macrophages (HMDMs) and their secretion of inflammatory chemokines are controlled by Rab5a. We find that C5a activation of the G protein coupled receptor C5aR1 expressed on the surface of HMDMs, recruits β-arrestin2 via Rab5a trafficking, then activates downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling that culminates in chemotaxis and secretion of pro-inflammatory chemokines from HMDMs. High-resolution lattice light-sheet microscopy on live cells showed that C5a activates C5aR1-GFP internalization and colocalization with Rab5a-tdTomato but not with dominant negative mutant Rab5a-S34N-tdTomato in HEK293 cells. We found that Rab5a is significantly upregulated in differentiated HMDMs and internalization of C5aR1 is dependent on Rab5a. Interestingly, while knockdown of Rab5a inhibited C5aR1-mediated Akt phosphorylation, it did not affect C5aR1-mediated ERK1/2 phosphorylation or intracellular calcium mobilization in HMDMs. Functional analysis using transwell migration and µ-slide chemotaxis assays indicated that Rab5a regulates C5a-induced chemotaxis of HMDMs. Further, C5aR1 was found to mediate interaction of Rab5a with β-arrestin2 but not with G proteins in HMDMs. Furthermore, C5a-induced secretion of pro-inflammatory chemokines (CCL2, CCL3) from HMDMs was attenuated by Rab5a or β-arrestin2 knockdown or by pharmacological inhibition with a C5aR1 antagonist or a PI3K inhibitor. These findings reveal a C5a-C5aR1-β-arrestin2-Rab5a-PI3K signaling pathway that regulates chemotaxis and pro-inflammatory chemokine secretion in HMDMs and suggests new ways of selectively modulating C5a-induced inflammatory outputs.