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Distinct types of immune responses are activated by infections, which cause the development of type I, II, or III inflammation, regulated by Th1, Th2, Th17 helper T cells and ILC1, ILC2 and ILC3 cells, respectively. While the classification of immune responses to different groups of pathogens is widely accepted, subtypes of the immune response elicited by sterile inflammation have not yet been detailed. Necroinflammation is associated with the release of damage-associated molecular patterns (DAMP) from dying cells. In this review, we present that the distinct molecular mechanisms activated during apoptosis, necroptosis, pyroptosis, and ferroptosis lead to the release of different patterns of DAMPs and their suppressors, SAMPs. We summarize the currently available data on how regulated cell death pathways and released DAMPs and SAMPs direct the differentiation of T helper and ILC cells. Understanding the subtypes of necroinflammation can be crucial in developing strategies for the treatment of sterile inflammatory diseases caused by cell death processes.

Most of the leading causes of death, such as cardiovascular diseases, cancer, dementia, neurodegenerative diseases, and many more, are associated with sterile inflammation, either as a cause or a consequence of these conditions. The ability to control the progression of inflammation toward tissue resolution before it becomes chronic holds significant clinical potential. During sterile inflammation, the initiation of inflammation occurs through damage-associated molecular patterns (DAMPs) in the absence of pathogen-associated molecules. Macrophages, which are primarily localized in the tissue, play a pivotal role in sensing DAMPs. Furthermore, macrophages can also detect and respond to resolution-associated molecular patterns (RAMPs) and specific pro-resolving mediators (SPMs) during sterile inflammation. Macrophages, being highly adaptable cells, are particularly influenced by changes in the microenvironment. In response to the tissue environment, monocytes, pro-inflammatory macrophages, and pro-resolution macrophages can modulate their differentiation state. Ultimately, DAMP and RAMP-primed macrophages, depending on the predominant subpopulation, regulate the balance between inflammatory and resolving processes. While sterile injury and pathogen-induced reactions may have distinct effects on macrophages, most studies have focused on macrophage responses induced by pathogens. In this review, which emphasizes available human data, we illustrate how macrophages sense these mediators by examining the expression of receptors for DAMPs, RAMPs, and SPMs. We also delve into the signaling pathways induced by DAMPs, RAMPs, and SPMs, which primarily contribute to the regulation of macrophage differentiation from a pro-inflammatory to a pro-resolution phenotype. Understanding the regulatory mechanisms behind the transition between macrophage subtypes can offer insights into manipulating the transition from inflammation to resolution in sterile inflammatory diseases.

Plasmacytoid dendritic cells (pDCs) are a unique subset of dendritic cells specialized in rapid and robust type I interferon (IFN) production, playing critical roles in the pathogenesis and pathomechanisms of many human diseases. Accurate identification of pDCs in peripheral blood mononuclear cells (PBMCs) is challenging due to dynamic and non-exclusive specific expression of surface markers such as blood dendritic cell antigen (BDCA)-2 and BDCA-4. Although BDCA-4 is generally more stably expressed than BDCA-2, prolonged stimulation or inflammatory conditions can induce its expression on multiple non-pDC cell types, reducing the accuracy of pDC identification. Here, we thoroughly investigated BDCA-4 expression dynamics on pDCs and other PBMC subsets following prolonged activation with Toll-like receptor (TLR) 7 and TLR9 agonists. Our flow cytometry analysis revealed a significant increase in BDCA-4-positive non-pDC populations after extended stimulation, primarily corresponding to CD14+ monocytes. To overcome this limitation, we performed a gating strategy combining BDCA-4 positivity with a cocktail of non-pDC markers, enabling the exclusion of non-pDCs and accurate identification of pDCs. This approach enables the reliable identification of pDCs within heterogeneous cell populations using only two fluorescent channels in healthy conditions and even during strong activation or pathological states characterized by chronic inflammation.

In addition to killing malignant cells, effective cancer therapies must also promote the development of an immunostimulatory tumor microenvironment (TME). Macrophages are the most abundant immune cell population within the TME. These highly plastic cells play key roles in tumor progression, chronic inflammation, immunosuppression, and metastasis. Although increasing research efforts focus on manipulating macrophage functions, relatively little is known about how standard anticancer strategies, especially chemotherapeutic agents, influence the composition, polarization state, and functional behavior of macrophage subpopulations. Chemotherapeutic agents remain a primary treatment option for many types of cancer, including breast and cervical cancers. In this study, we used epirubicin and doxorubicin at near-therapeutic concentrations and examined their effects on macrophage functions in co-culture with MDA-MB-231 breast cancer and HeLa cervical cancer cell lines. We demonstrated that the presence of tumor cells led to increased expression of the M2 macrophage marker CD206, a change that was reduced by both chemotherapeutic agents. The production of macrophage-derived chemokines, such as IP-10 and IL-8, was also altered by tumor presence and drug exposure. A striking finding was that the co-presence of chemotherapeutic agents and MDA-MB-231 cells synergistically altered macrophage motility. This effect was not observed in monocultures. Furthermore, the presence of tumor cells reduced the susceptibility of pro-inflammatory M1 macrophages to drug-induced cell death. These results indicate that chemotherapy can reshape the macrophage landscape in the TME. We highlight that the combined effects of tumor cell presence and chemotherapy modulate the composition, phenotype, and migration of macrophage subtypes differently than either factor alone.

IntroductionExtracts and compounds isolated from hemp (Cannabis sativa) are increasingly gaining popularity in the treatment of a number of diseases, with topical formulations for dermatological conditions leading the way. Phytocannabinoids such as ( )-cannabidiol, ( )-cannabinol and ( )-Δ9-tetrahydrocannabivarin (CBD, CBN, and THCV, respectively), are present in variable amounts in the plant, and have been shown to have mostly anti-inflammatory effects both in vitro and in vivo, albeit dominantly in murine models. The role of phytocannabinoids in regulating responses of dendritic cells (DCs) remains unclear.MethodsOur research aimed to investigate the effects of CBD, CBN, and THCV on human DCs differentiated from monocytes (moDCs). moDCs were treated with up to 10 μM of each phytocannabinoid, and their effects on viability, differentiation, and maturation were assessed both alone, and in conjunction with TLR agonists. The effects of CBD on cytokine production, T cell activation and polarization as well as the transcriptome of moDCs was also determined.ResultsPhytocannabinoids did not influence the viability of moDCs up to 10 μM, and only CBD had effects on maturational markers of moDCs, and neither compound influenced LPS-induced activation at 10 μM. Since only CBD had measurable effects on moDCs, in our subsequent experiments we tested the effect only of that pCB. On moDCs differentiated in the presence of CBD subsequent activation by LPS induced a markedly different, much more tolerogenic response. CBD-treated moDCs also produced significantly more interleukin (IL)-6, TNFα and, importantly, IL-10 in response to LPS, which shows a shift toward anti-inflammatory signaling, as well as a more robust secretory response in general. To rule out the possibility that these effects of CBD are specific to TLR4 signaling, we determined the effect of CBD on TLR7/8-induced maturation as well, and saw similar, although less marked responses. CBD-treated moDCs were also less efficient at activating naïve T cells after LPS stimulation, further supporting the tolerogenic effect of this phytocannabinoid on moDCs. Reactome pathway analysis showed an inflammatory response to LPS in moDCs, and to a lesser extent to CBD as well. In contrast CBD-treated moDCs responded to LPS with a shift towards a more tolerogenic phenotype, as IL-10 signaling was the most prominently induced pathway in this group.DiscussionOur results show that CBD achieves an anti-inflammatory effect on adaptive immune responses only in the presence of an activating stimuli on moDCs by reprogramming cells during long-term treatment, and not through acute, short-term effects.

The endocannabinoid system (ECS), named after the chemical compounds found in the cannabis plant, is a regulatory network of neurotransmitters, receptors, and enzymes that plays crucial roles in skin health and disease. Endogenous ligands of the ECS, called endocannabinoids, have proven to be important regulators of immune responses. One of the most prevalent endocannabinoids, arachidonoylethanolamide (also known as anandamide), is known for its anti-inflammatory effects. Langerhans cells (LCs) are the sole antigen-presenting cells present in the human epidermis. They serve as the first line of defense against pathogens and are essential for the skin's specific immune responses and play a critical role in maintaining tissue homeostasis; however, little is known about the effect of endocannabinoids on these cells. Our research aimed to provide the connection between monocyte-derived Langerhans cells (moLCs) and the ECS, shedding light on their collaborative roles in immune homeostasis and inflammation. Human monocytes were differentiated into moLCs using established protocols. Anandamide was applied during the differentiation process to test its effect on the viability, marker expression, and cytokine production of the cells, as well as in short term treatments for intracellular calcium measurement. TLR ligands applied after the differentiation protocol were used to activate moLCs. The impact of anandamide on the functionality of moLCs was further assessed using differential gene expression analysis of bulk RNA-Seq data, moLC-T cell cocultures, while ELISpot was employed to determine polarization of T cells activated in the aforementioned cocultures. Anandamide did not significantly affect the viability of moLCs up to 10 µM. When applied during the differentiation process it had only a negligible effect on CD207 expression, the prototypic marker of LCs; however, there was an observed reduction in CD1a expression by moLCs. Anandamide had no significant effects on the maturation status of moLCs, nor did it affect the maturation induced by TLR3 and TLR7/8 agonists. MoLCs differentiated in the presence of anandamide did however show decreased production of CXCL8, IL-6, IL-10 and IL-12 cytokines induced by TLR3 and TLR7/8 activation. Anandamide-treated moLCs showed an increased capability to activate naïve T cells; however, not to the level seen with combined TLR agonism. RNA sequencing analysis of moLCs differentiated with anandamide showed modest changes compared to control cells but did reveal an inhibitory effect on oxidative phosphorylation specifically in activated moLCs. Anandamide also promoted the polarization of naïve T cells towards a Th1 phenotype. Our results show that anandamide has nuanced effects on the differentiation, maturation, cytokine secretion, metabolism and function of activated moLCs. Among these changes the decrease in CD1a expression on moLCs holds promise to selectively dampen inflammation induced by CD1a restricted T cells, which have been implicated as drivers of inflammation in common inflammatory skin conditions such as psoriasis, atopic dermatitis and contact dermatitis.

The interaction between the nervous and immune systems is crucial for maintaining homeostasis and can influence disease progression in inflammatory skin diseases, such as atopic dermatitis (AD). Sensory neurons in the skin can secrete neuropeptides that modulate immune cell activity, including Langerhans cells (LCs), one of the primary antigen-presenting cells in the epidermis. In our study we investigated the effects of neuropeptides on the differentiation of monocyte-derived LCs (moLCs), specifically the neuropeptides with the most profound effect, i.e. atrial- and B-type natriuretic peptides (ANP and BNP, respectively). RNA sequencing and RT-qPCR were used to analyze neuropeptide receptor expression in moLCs and immature dendritic cells (iDCs), and the most translationally relevant, natriuretic peptide receptor A (NPR1) was validated on the protein level using western blotting. Cell surface markers of moLCs were assessed using flow cytometry, and NPR1 functionality was confirmed through intracellular cGMP assays. Confocal microscopy was used to confirm the expression of NPR1 in healthy and AD skin. RNA-Seq analysis was also employed to characterize the phenotypic changes in moLCs differentiated in the presence of BNP. NPR1 expression was significantly higher in moLCs compared to iDCs, and treatment with ANP and BNP enhanced moLC differentiation, increasing CD207, CD1a, and HLA-DQ expression, while other tested neuropeptides (calcitonin gene-related peptide [CGRP], neurotensin) had no significant effect. NPR1 was functionally active, as evidenced by increased intracellular cGMP levels upon ligand binding. Confocal microscopy revealed NPR1 expression on LC cell bodies in both healthy and AD skin, with reduced intensity in AD. RNA-Seq analysis of BNP-treated moLCs indicated a shift toward a migratory LC phenotype, marked by upregulation of genes associated with cell motility (e.g., CCR7, LAMP3). These findings demonstrate that NPR1 activation promotes a migratory LC phenotype, highlighting the role of neuropeptides in shaping cutaneous immune responses. The reduced number of LCs in AD skin suggests a potential link between neuropeptide signaling and disease pathology.

Chronic diseases affecting the cardiovascular system, diabetes mellitus, neurodegenerative diseases, and various other organ-specific conditions, involve different underlying pathological processes. However, they share common risk factors that contribute to the development and progression of these diseases, including air pollution, hypertension, obesity, high cholesterol levels, smoking and alcoholism. In this review, we aim to explore the connection between four types of diseases with different etiologies and various risk factors. We highlight that the presence of risk factors induces regulated necrotic cell death, leading to the release of damage-associated molecular patterns (DAMPs), ultimately resulting in sterile inflammation. Therefore, DAMP-mediated inflammation may be the link explaining how risk factors can lead to the development and maintenance of chronic diseases. To explore these processes, we summarize the main cell death pathways activated by the most common life-threatening risk factors, the types of released DAMPs and how these events are associated with the pathophysiology of diseases with the highest mortality. Various risk factors, such as smoking, air pollution, alcoholism, hypertension, obesity, and high cholesterol levels induce regulated necrosis. Subsequently, the release of DAMPs leads to chronic inflammation, which increases the risk of many diseases, including those with the highest mortality rates.

Cancers reprogram macrophages (MΦs) to a tumor-growth-promoting TAM (tumor-associated MΦ) phenotype that is similar to the anti-inflammatory M2 phenotype. Poly(ADP-ribose) polymerase (PARP) enzymes regulate various aspects of MΦ biology, but their role in the development of TAM phenotype has not yet been investigated. Here, we show that the multispectral PARP inhibitor (PARPi) PJ34 and the PARP14 specific inhibitor MCD113 suppress the expression of M2 marker genes in IL-4-polarized primary murine MΦs, in THP-1 monocytic human MΦs, and in primary human monocyte-derived MΦs. MΦs isolated from PARP14 knockout mice showed a limited ability to differentiate to M2 cells. In a murine model of TAM polarization (4T1 breast carcinoma cell supernatant transfer to primary MΦs) and in a human TAM model (spheroids formed from JIMT-1 breast carcinoma cells and THP-1-MΦs), both PARPis and the PARP14 KO phenotype caused weaker TAM polarization. Increased JIMT-1 cell apoptosis in co-culture spheroids treated with PARPis suggested reduced functional TAM reprogramming. Protein profiling arrays identified lipocalin-2, macrophage migration inhibitory factor, and plasminogen activator inhibitor-1 as potential (ADP-ribosyl)ation-dependent mediators of TAM differentiation. Our data suggest that PARP14 inhibition might be a viable anticancer strategy with a potential to boost anticancer immune responses by reprogramming TAMs.

Developing dendritic cells (DCs) from monocytes is a sensitively regulated process. One possible way for cancers to avoid immune recognition and antitumor response is the modulation of DC differentiation. Although several studies are available on the examination of tumor-associated macrophages, a comprehensive analysis focusing on the effects of tumor-formed DCs is not known to date. We provide a comparative analysis of the tumor-edited-monocyte derived DCs differentiated in the presence of adenocarcinomas (MDA, HT29, HeLa)- and primary (WM278, WM983A) or metastatic (WM1617, WM983B) melanomas. The immunomodulatory effect of tumors is mediated at least partly by secreted mediators. We investigated the impact of tumor cell-derived conditioned media on the differentiation of DCs from CD14 + monocytes, sequentially determining the phenotype, cytokine production, phagocytic, and the T cell polarizing capacity of moDCs. We completed our observations by analyzing our data with bioinformatic tools to provide objective correlations between phenotypical and functional properties of different tumor-educated moDCs. The correlation analysis revealed significant differences in the characteristics of adenocarcinomas- or melanomas-edited moDCs. We highlight the functional differences in the properties of moDCs differentiated in the presence of various cancer cell lines. We offer new information and options for the in vitro differentiation protocols of various tumor-conditioned moDCs. Our results confirm that various immunomodulatory properties of different tumor cell lines result in multiple manipulations of DC differentiation.