Macrophage Migration Research Articles

Macrophage migration is differentially regulated by fibronectin and laminin through altered adhesion and myosin II localization.

Macrophages are indispensable for proper immune surveillance and inflammatory regulation. They also exhibit dramatic phenotypic plasticity and are highly responsive to their local microenvironment, which includes the extracellular matrix (ECM). This work demonstrates that two fibrous ECM glycoproteins, fibronectin (FN) and laminin (LAM), elicit distinct morphological and migratory responses from macrophages in two-dimensional environments. LAM 111 inhibits macrophage cell spreading, but drives them to migrate rapidly and less persistently compared with cells on FN. Differential integrin engagement and ROCK/myosin II organization helps explain why macrophages alter their morphology and migration character on these two ECM components. This study also demonstrates that LAM 111 exerts a suppressive effect toward FN, as macrophages plated on a LAM/FN mixture adopt a morphology and migratory character almost identical to LAM alone. This suggests that distinct responses can be initiated downstream of receptor-ECM engagement, and that one component of the microenvironment may affect the cell's ability to sense another. Overall, macrophages appear intrinsically poised to rapidly switch between distinct migratory characters based on their ECM environments. The role of ECM composition in dictating motile and inflammatory responses in three-dimensional and in vivo contexts warrants further study.

High species homology potentiates quantitative inflammation profiling in zebrafish using immunofluorescence

Due to substantial homology between the human and zebrafish genome and a high level of conservation of the innate immune system across species, zebrafish larvae have become an invaluable research tool for studying inflammation and modelling inflammatory disease. However, further microscopy techniques need to be developed for better profiling of inflammation and in particular, integrated cytokine responses to different stimuli - approaches are currently largely limited to assessment of changes in cytokine gene transcription and in vivo visualisation using transgenics, which is limited in terms of the number of cytokines that may be assessed at once. In this study, after confirming substantial homology of human vs zebrafish cytokine amino acid sequences, immunofluorescence staining using antibodies directed at human cytokines was performed. Inflammatory cytokine signalling responses to experimental tailfin transection was assessed over 24 h (1 hpi (hours post injury), 2 hpi, 4 hpi, 24 hpi) in zebrafish larvae, with experimental end point at 120 h post fertilization (hpf). When immunofluorescence results were compared to responses observed in rodent and human literature, it is clear that the cytokines follow a similar response, albeit with a condensed total time course. Notably, tumor necrosis factor-α and monocyte chemoattractant protein-1 increased and remained elevated over the 24-h period. In contrast, interleukin-1β and interleukin-6 peaked at 4 hpi and 2 hpi respectively but had both returned to baseline levels by 24 hpi. Macrophage migration inhibitory factor was lowest at 1 hpi, potentially encouraging macrophage movement into the site of injury, followed by a sharp increase. This protocol provides valuable insight into inflammation over a time course and more so, provides an affordable and accessible method to comprehensively assess inflammation in zebrafish disease models.

Leukotriene B4 receptor 2 governs macrophage migration during tissue inflammation

Chronic inflammation is the underlying cause of many diseases, including type 1 diabetes, obesity, and non-alcoholic fatty liver disease. Macrophages are continuously recruited to tissues during chronic inflammation where they exacerbate or resolve the pro-inflammatory environment. Although leukotriene B4 receptor 2 (BLT2) has been characterized as a low affinity receptor to several key eicosanoids and chemoattractants, its precise roles in the setting of inflammation and macrophage function remain incompletely understood. Here we used zebrafish and mouse models to probe the role of BLT2 in macrophage function during inflammation. We detected BLT2 expression in bone marrow derived and peritoneal macrophages of mouse models. Transcriptomic analysis of Ltb4r2-/- and WT macrophages suggested a role for BLT2 in macrophage migration, and studies invitro confirmed that whereas BLT2 does not mediate macrophage polarization, it is required for chemotactic function, possibly mediated by downstream genes Ccl5 and Lgals3. Using a zebrafish model of tailfin injury, we demonstrated that antisense morpholino-mediated knockdown of blt2a or chemical inhibition of BLT2 signaling impairs macrophage migration. We further replicated these findings in zebrafish models of islet injury and liver inflammation. Moreover, we established the applicability of our zebrafish findings to mammals by showing that macrophages of Ltb4r2-/- mice have defective migration during lipopolysaccharide stimulation invivo. Collectively, our results demonstrate that BLT2 mediates macrophage migration during inflammation, which implicates it as a potential therapeutic target for inflammatory pathologies.

Neighborhood racial composition and experiences of racial discrimination: Associations with cytokines during pregnancy among African American women

BackgroundPreterm birth rates are consistently higher in African American (AA) pregnancies compared to White pregnancies in the United States. Neighborhood racial composition, experiences of racial discrimination, and systemic inflammation are factors that have been associated with preterm birth and other adverse pregnancy outcomes that may account for these disparities. Here, we investigated whether perceived neighborhood racial composition and experiences of discrimination were predictive of cytokine levels during pregnancy among AA individuals. Methods545 AA individuals completed surveys and had blood samples collected at prenatal clinics in the Midwest at three timepoints (8–18,19-29, and 30–36 weeks gestation) throughout pregnancy. Pro-inflammatory [interferon (IFN)-γ, interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, macrophage migration inhibitory factor (MIF)] and anti-inflammatory cytokines (IL-10) were quantified. Multivariate and multilevel models were used to examine associations of perceived neighborhood racial composition and experiences of racial discrimination with cytokine levels, controlling for relevant covariates. ResultsPerceived neighborhood racial composition was significantly associated with MIF at 30–36 weeks gestation in multivariate regression (p < 0.001). Living in neighborhoods with more compared to fewer White people was predictive of higher levels of MIF (b = 0.599, SE = 0.12, p < 0.001). Experiences of discrimination were also associated with higher levels of MIF (β = 0.141, SE = 0.07, p = 0.036). Neither predictor was associated with other cytokines. Follow-up analyses revealed that neighborhood racial composition was also predictive of higher MIF levels at 8–18 weeks gestation (p = 0.02) and at 19–29 weeks gestation (p = 0.04). ConclusionsLiving in neighborhoods with more White individuals and having more lifetime experiences of racial discrimination were positively related to levels of the pro-inflammatory cytokine, MIF, among pregnant AA individuals. MIF’s known positive relationships with chronic stress and preterm birth suggest that these elevations in MIF may have negative health consequences. Future studies should explore whether MIF serves as a pathway between neighborhood racial composition or experiences of racial discrimination and preterm birth risk among AA individuals.

Low-Dose Trypsin Accelerates Wound Healing via Protease-Activated Receptor 2.

The management of wounds remains a significant healthcare challenge, highlighting the need for effective wound healing strategies. To address this, it is crucial to explore the molecular mechanisms underlying tissue repair as well as explore potential therapeutic approaches. Trypsin, as a serine protease, has been clinically utilized for wound healing for decades; however, it still lacks systemic investigation on its role and related mechanism. This study aimed to investigate the effects of low-dose trypsin on wound healing both in vitro and in vivo. While trypsin is an endogenous stimulus for protease-activated receptor 2 (PAR2), we discovered that both low-dose trypsin and synthesized PAR2 agonists significantly enhanced the migration, adhesion, and proliferation of fibroblasts and macrophages, similar to the natural repair mechanism mediated by mast cell tryptase. Moreover, such cell functions induced by trypsin were largely inhibited by PAR2 blockade, indicating the participation of trypsin via PAR2 activation. Additionally, low-dose trypsin notably expedited healing and regeneration while enhancing collagen deposition in skin wounds in vivo. Importantly, upon stimulation of trypsin or PAR2 agonists, there were significant upregulations of genes including claudin-7 (Cldn7), occludin (Ocln), and interleukin-17A (IL-17A) associated with proliferation and migration, extracellular matrix (ECM), tight junction, and focal adhesion, which contributed to wound healing. In summary, our study suggested that a low-dose trypsin could be a promising strategy for wound healing, and its function was highly dependent on PAR2 activation.

Effect of animal venom toxins on the main links of the homeostasis of mammals (Review).

The human body is affected by environmental factors. The dynamic balance between the organism and its environment results from the influence of natural, anthropogenic and social aspects. The factors of exogenous origin determine development of adaptive changes. The present article summarises the mechanisms of animal venom toxins and homeostasis disruption in the body of mammals. The mechanisms underlying pathological changes are associated with shifts in biochemical reactions. Components of the immune, nervous and endocrine systems are key in the host defense and adaptation processes in response to venom by triggering signalling pathways (PI3kinase pathway, arachidonic acid cascade). Animal venom toxins initiate the development of inflammatory processes, the synthesis of pro-inflammatory mediators (cytokines), ROS, proteolytic enzymes, activate the migration of leukocytes and macrophages. Keratinocytes and endothelial cells act as protective barriers under the action of animal venom toxins on the body of mammals. In addition, the formation of pores in cell membranes, structural changes in cell ion channels are characteristic of the action of animal venom toxins.

Design, synthesis, and evaluation of pirfenidone-NSAIDs conjugates for the treatment of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal, chronic and progressive lung disease that threaten public health like many cancers. In this study, targeting the significant driving factor, inflammatory response, of the IPF, several conjugates of pirfenidone (PFD) with non-steroidal anti-inflammatory drugs (NSAIDs), along with their derivatives, were designed and synthesized to enhance the anti-IPF potency of PFD. Among these compounds, the (S)-ibuprofen-PFD conjugate 5b exhibited the most potent anti-proliferation activity against NIH3T3 cells, demonstrating up to a 343-fold improvement compared to PFD (IC50 = 0.04 mM vs IC50 = 13.72 mM). Notably, 5b exhibited superior activity in inhibiting the migration of macrophages induced by TGF-β compared to PFD. Additionally, 5b demonstrated significant suppression of TGF-β-induced migration of NIH3T3 cells and induction of apoptosis in NIH3T3 cells. Mechanistic studies revealed that 5b reduced the expression of collagen I and α-SMA by inhibiting the TGF-β/SMAD3 pathway. In a bleomycin-induced pulmonary fibrosis model, treatment with 5b (40 mg/kg/day, orally) exhibited a more pronounced effect on reducing the degree of histopathological changes in lung tissue and alleviating collagen deposition compared to PFD (100 mg/kg/day, orally). Moreover, 5b could block the expression of collagen I, α-SMA, fibronectin, and pro-inflammatory factors (IL-6, IFN-γ, and TNF-α) compared to PFD, while demonstrating low toxicity in vivo. These preliminary results indicated that the hybridization of PFD with NSAIDs represented an effective modification approach to improve the anti-IPF potency of PFD. Consequently, 5b emerged as a promising candidate for the further development of new anti-IPF agents.

Bisdemethoxycurcumin, a curcumin derivative, ameliorates adjuvant-induced arthritis by suppressing inflammatory reactions and macrophage migration

Rheumatoid arthritis (RA) is a highly prevalent and chronic inflammatory synovial joint disease manifested by hyperplasia and continuous inflammation. Curcumin (Cur) has been studied for alleviating RA. However, poor stability and oral bioavailability restrict its therapeutic value. Bisdemethoxycurcumin (BDMC), a curcumin (Cur) derivative, exerts better stability and oral bioavailability than Cur. However, the efficacy of BDMC on RA has not been fully clarified. The aim of the study was to investigate the therapeutic effects and underlying mechanisms of BDMC on RA. The in-vivo anti-arthritic activity of BDMC was determined via adjuvant-induced arthritis (AIA) rat model. Paw swelling, body weight, arthritic index, and histopathological assessments were performed. RAW264.7 cell was stimulated by lipopolysaccharides (LPS) in vitro. The cell viability were determined by CCK8 assay, while the migration ability was determined using cell wound healing and transwell assays. Furthermore, in-vivo and in-vitro levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were assayed by ELISA, and that of IκBα, p-NF-κB, NF-κB, and COX-2 were assessed via Western blot or immunofluorescence. In AIA rat model, it suggested a higher anti-arthritic activity of BDMC than Cur, including amelioration of swelling in hind paws, reduced arthritic index, and alleviated histopathological injury in rats. Furthermore, BDMC also substantially decreased the levels of the aforementioned pro-inflammatory cytokines in both in-vivo and in-vitro, inhibited the IκBα degradation, down-regulated the COX-2 levels and p-NF-κB/NF-κB ratio in AIA rats and LPS-stimulated RAW264.7 cells. Additionally, BDMC showed an inhibitory effect on the migration of LPS-stimulated RAW264.7 cells. BDMC could effectively ameliorate RA by suppressing inflammatory reactions and inhibiting macrophage migration, more potentially than Cur.

Expression of Concern: Tanshinone IIA suppresses the progression of atherosclerosis by inhibiting the apoptosis of vascular smooth muscle cells and the proliferation and migration of macrophages induced by ox-LDL.

Expression of Concern: Tanshinone IIA suppresses the progression of atherosclerosis by inhibiting the apoptosis of vascular smooth muscle cells and the proliferation and migration of macrophages induced by ox-LDL.

Phosphorylation of MIF by PIP4K2a is necessary for cilia biogenesis

Primary cilia are microtubule-based organelles that play important roles in development and tissue homeostasis. Macrophage migration inhibitory factor (MIF) has long been recognized as a secreted cytokine in the pathogenesis of various human diseases, including cancer and autosomal dominant polycystic kidney disease (ADPKD). Unlike other cytokines, unique functional characteristics of intracellular MIF have emerged. In this study, we show that MIF is localized and formed a ring like structure at the proximal end of centrioles, where it regulates cilia biogenesis through affecting 1) the recruitment of TTBK2 to basal body and the removal of CP110 from mother centriole, 2) the accumulation of CEP290 at centriolar satellites, and 3) the trafficking of intraflagellar transport (IFT) related proteins. We also show that MIF functions as a novel transcriptional factor to regulate the expression of genes related to ciliogenesis via binding on the promotors of those genes. MIF also binds chromatin and regulates transcription of genes involved in diverse homeostatic signaling pathways. We identify phosphatidylinositol-5-phosphate 4-kinase type 2 alpha (PIP4K2a) as an upstream regulator of MIF, which interacts with and phosphorylates MIF at S91 to increase its interaction with 14-3-3ζ, resulting in its nuclear translocation and transcription regulation. This study suggests that MIF is a key player in cilia biogenesis and a novel transcriptional regulator in homeostasis, which forward our understanding of how MIF is able to carry out several nonoverlapping functions.

A carboxymethyl chitosan/oxidized hyaluronic acid composite hydrogel dressing loading with stem cell exosome for chronic inflammation wounds healing

Stem cell exosomes (Exo) play an important role in the transformation of macrophages, but the rapid clearance of Exo in vivo limits their therapeutic effects for chronic inflammation wounds healing. Here, stem cell Exo was isolated and introduced to a composite hydrogel including carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (OHA) through chemical cross-linking, which formed an Exo-loaded (CMCS/OHA/Exo) hydrogel. The CMCS/OHA/Exo hydrogel exhibited a function of Exo sustained release and an Exo protection within 6 days. This CMCS/OHA/Exo hydrogel was much better than CMCS/OHA hydrogel or Exo solution in macrophage cell phagocytosis, proliferation and migration in vitro, especially, played an obviously positive role in the transformation of macrophages compared with the reference groups. For the treatment of the chronic inflammation wounds in vivo, the CMCS/OHA/Exo hydrogel had the best results at wound heal rate and inhibiting the secretion of inflammatory factors, and it was far superior to reference groups in wound re-epithelization and collagen production. CMCS/OHA/Exo hydrogels can promote Exo release based on hydrogel degradation to regulate macrophages transformation and accelerate chronic wound healing. The study offers a method for preparing Exo-loaded hydrogels that effectively promote the transformation of macrophages and accelerate chronic inflammatory wound healing.

DRhQ: a novel therapeutic that improves cognition and mitochondrial function and reduces neuroinflammation in beta‐amyloid overexpressing Mice

AbstractBackgroundNeuroinflammation and mitochondrial dysfunction are central to the progression of Alzheimer’s disease (AD), provoking neuronal loss and cognitive decline. Identifying strategies for mitigating these responses could therefore be of significant therapeutic relevance. Evidence suggests that the inflammatory axis mediated by macrophage migration inhibitory factory (MIF) binding to its receptor, CD74, plays an important role in many central nervous system (CNS) disorders like AD. Our group has developed DRhQ, a novel CD74 binding construct that competitively inhibits MIF binding, blocks T‐cell and macrophage activation and migration into the CNS, enhances anti‐inflammatory microglia cell numbers and reduces pro‐inflammatory gene expression. DRhQ treatment resulted in reduced neuroinflammation and disease severity in a mouse model of multiple sclerosis. Because neuroinflammation is a prominent in the AD brain as well we hypothesized that DRhQ could be a similarly effective neuroprotective agent in the context of AD. We evaluated this hypothesis using the 5xFAD mouse model of β‐amyloid (Aβ) accumulation.MethodSix month old male and female 5xFAD mice and their wild‐type littermates will be treated with 0 or 100 ug of DRhQ subcutaneously for five days followed by a 3 day booster series weekly for the next 3 weeks. During the 4th week of treatment mice were evaluated cognitively using the novel object recognition test and brain tissue was harvested to evaluate mitochondrial function, Aβ pathology as well as markers of microglial activation and neuroinflammation.ResultDRhQ improved cognition and reduced neuroinflammatory markers in both male and female 5xFAD mice. DRhQ also significantly attenuated deficits in cortical mitochondrial function in female 5xFAD mice and elicited a similar, though non‐significant, effect in male 5xFAD mice. Aβ plaque burden was likewise reduced in the hippocampus and cortex of female 5xFAD mice but not significantly reduced in male 5xFAD animals.ConclusionThese results suggest that DRhQ attenuates Aβ pathology, mitochondrial dysfunction and neuroinflammation and improves cognitive function in 5xFAD mice. Future experiments will explore dose response effects and investigate the apparent sex differences in the magnitude of effect of DRhQ on specific endpoints in these mice as well as evaluate the effects of DRhQ in models of tauopathy.

Synergistically Enhancing Immunotherapy Efficacy in Glioblastoma with Gold-Core Silica-Shell Nanoparticles and Radiation.

Glioblastoma is a highly aggressive brain tumor with universally poor outcomes. Recent progress in immune checkpoint inhibitors has led to increased interest in their application in glioblastoma. Nonetheless, the unique immune milieu in the brain has posed remarkable challenges to the efficacy of immunotherapy. We aimed to leverage the radiation-induced immunogenic cell death to overcome the immunosuppressive network in glioblastoma. We developed a novel approach using the gold-core silica-shell nanoparticles (Au@SiO2 NPs) in combination with low-dose radiation to enhance the therapeutic efficacy of the immune checkpoint inhibitor (atezolizumab) in brain tumors. The biocompatibility, immune cell recruitment, and antitumor ability of the combinatorial strategy were determined using in vitro assays and in vivo models. Our approach successfully induced the migration of macrophages towards brain tumors and promoted cancer cell apoptosis. Subcutaneous tumor models demonstrated favorable safety profiles and significantly enhanced anticancer effects. In orthotopic brain tumor models, the multimodal therapy yielded substantial prognostic benefits over any individual modalities, achieving an impressive 40% survival rate. In summary, the combination of Au@SiO2 NPs and low-dose radiation holds the potential to improve the clinical efficacy of immune checkpoint inhibitors. The synergetic strategy modulates tumor microenvironments and enhances systemic antitumor immunity, paving a novel way for glioblastoma treatment.

Single-cell transcriptomics reveals ferrimagnetic vortex iron oxide nanoring-mediated mild magnetic hyperthermia exerts antitumor effects by alleviating macrophage suppression in breast cancer

The potential of Ferrimagnetic vortex iron oxide nanoring-mediated mild magnetic hyperthermia (FVIO-MHT) in solid tumor therapy has been demonstrated. However, the impact of FVIO-MHT on the tumor microenvironment (TME) remains unclear. This study utilized single-cell transcriptome sequencing to examine the alterations in the TME in response to FVIO-MHT in breast cancer. The results revealed the cellular composition within the tumor microenvironment (TME) was primarily modified due to a decrease in tumor cells and an increased infiltration of myeloid cells. Subsequently, an enhancement in active oxygen (ROS) metabolism was observed, indicating oxidative damage to tumor cells. Interestingly, FVIO-MHT reprogrammed the macrophages’ phenotypes, as evidenced by alterations in the transcriptome characteristics associated with both classic and alternative activated phenotypes. And an elevated level of ROS generation and oxidative phosphorylation suggested that activated phagocytosis and inflammation occurred in macrophages. Additionally, cell–cell communication analysis revealed that FVIO-MHT attenuated the suppression between tumor cells and macrophages by inhibiting phagocytic checkpoint and macrophage migration inhibitory factor signaling pathways. Inhibition of B2m, an anti-phagocytosis checkpoint, could promote macrophage-mediated phagocytosis and significantly inhibit tumor growth. These data emphasize FVIO-MHT may promote the antitumor capabilities of macrophages by alleviating the suppression between tumor cells and macrophages.

TRAF3 gene regulates macrophage migration and activation by lung epithelial cells infected with Aspergillus fumigatus.

Aspergillus fumigatus can infect immunocompromised individuals and cause chronic and fatal invasive fungal infections. A better understanding of the molecular mechanisms of A. fumigatus-host interactions may provide new references for disease treatment. In this study, we demonstrated that the TRAF3 gene plays an important role in the early infection of A. fumigatus by regulating the resistance of lung epithelial cells to A. fumigatus. Macrophages are the most abundant innate immune cells in the alveoli; however, few studies have reported on the interactions between lung epithelial cells and macrophages in response to A. fumigatus invasion. In our study, it was demonstrated that the TRAF3 gene reduces migration to macrophages and cytokine production by negatively regulating lung epithelial cell adhesion and internalization of A. fumigatus spores. Together, our results provide new insights into lung epithelial cell-macrophage interactions during A. fumigatus infection.

The Role of Macrophage Migration Inhibitory Factor (MIF) in Regulation of T-Cell Activity in Multiple Myeloma

Background: Multiple myeloma (MM) is the second most common hematological malignancy associated with the overproduction of light chain or monoclonal immunoglobulin. MM remains incurable, with high relapses and refractory disease rates after first-line treatment. Hence, novel therapeutic approaches are needed to improve immune responses to prevent relapses or drug resistance after initial therapies. Immune responses are shaped by several factors, including mutations, pro- and anti-inflammatory cytokines and chemokines, and activation of immune checkpoints in the tumor microenvironment (TME) (Gonzalez et al., 2018). Macrophage migration inhibitory factor (MIF) as a soluble pro-inflammatory cytokine has become one of the most puzzling regulators of innate and adaptive immune responses. In myeloma, MIF was found to be significantly higher in MM cells from relapsed patients than those responding to the treatment. MIF has been implicated in drug resistance and suggested as a biomarker for predicting MM patient responses to proteasome inhibitors (Wang et al., 2020). We previously reported that MIF is highly expressed in MM and regulates CD84 (SLAMF5) expression in MM TME. We showed that MIF secreted from MM cells regulates PD-L1 expression through CD84 on CD14 + monocytic cells in the TME. Inhibition of MIF significantly reduced CD84 and PD-L1 expressions on human CD14 + cells and on myeloid-derived suppressor cells of the mice bearing MM (Lewinsky and Gunes et al., 2021). However, whether MIF directly regulates T-cell activity in MM TME has yet to be explored. Methods and Results: The current study reports that MIF may play a pivotal role in T-cell exhaustion in MM. Ouranalyses in published MM transcriptomic databases show that MM patients with low MIF had significantly longer overall and post-progression survival, indicating the therapeutic potential of MIF in MM. We also noted upregulated MIF gene expression in relapsed or refractory MM (R/RMM) patients (n=2) compared to that in newly diagnosed MM patients (n=7) and healthy individuals (n=9) in our and publicly available MM single-cell RNA sequencing datasets. We detected a profound MIF expression in MM cells and myeloid cell fractions, including monocytes, macrophages, and dendritic cells from the patients' bone marrow (BM) specimens. We previously reported that human MM cell lines highly express MIF. In this study, we assessed the impact of MIF secreted by MM cells on the immune cells, specifically CD8 + T cells. Our results showed that inhibition of MIF reduced the elevated T-cell exhaustion markers, PD1 and CTLA-4, on CD8 + T cells in healthy donors (HDs) peripheral blood mononuclear cells (PBMCs) following the coculturing with MM1.S cells (n=3 *P &amp;lt; 0.05 ). We also found that MIF inhibition induced the T-cell activation marker, LAMP1, on CD8 + T cells in this coculturing setting (n=3, **P &amp;lt; 0.01). Furthermore, we noted MIF inhibition reduced PD1 expression on CD8 + T cells in HDs PBMCs (n=3, **P &amp;lt; 0.01), suggesting MIF may regulate the exhaustion profile of CD8 + T cells. MIF can conduct paracrine or autocrine signalings by binding to its receptors, including CD74 and CD44, and lead to intracellular signaling cascades. Hence, we assessed CD74 gene expression as the counterpart of MIF in R/RMM, MM, and healthy patients. We noted significantly upregulated CD74 expression in R/RMM patients compared to newly diagnosed MM patients and healthy individuals. We also noted higher CD74 expression in CD8 + T cells than in CD4 + T cells in R/RMM patients, suggesting that MIF/CD74 axis may be a potential target to enhance CD8 + T cell-mediated immunity in R/RMM. To further explore the impact of MIF inhibition on MM progression, we performed a pilot in vivo study by inoculating murine 5TGM1 cells into two groups of syngeneic C57BL/KaLwRijHsd mice. After two weeks, the mice were injected with either sterile PBS as a control or a MIF inhibitor. The results of this pilot study showed that MIF inhibition significantly decreased MM cells in BM and PB of myeloma-bearing mice and reduced T-cell exhaustion markers on CD8 + T cells compared to that in the control mice (n=3-4, *P &amp;lt; 0.05, **P &amp;lt; 0.01, ***P &amp;lt; 0.001). Conclusion: Overall, our preliminary data indicate that MIF may have a crucial role in T-cell activity by regulating T-cell exhaustion markers in MM. Further studies are needed to explore the therapeutic potential of MIF to reduce T-cell exhaustion to improve anti-tumor immunity in MM.

Active STAT3 Prevents Selinexor-Induced Caspase-Independent Apoptosis in Multiple Myeloma

Multiple myeloma (MM) is an acquired malignant plasma cell disorder that typically develops late in life with a median age at diagnosis of 69 years and results in approximately 12,000 deaths per year. Currently, the standard treatment strategy for MM is a combination of 3 drugs with different mechanisms: dexamethasone, a proteasome inhibitor, and an immunomodulatory agent. Many MM patients acquire a “double-hit” of their TP53 on Chromosome 17p region after multple rounds of treatment and become resistant to these conventional therapeutics. Thus, novel effective treatments for high-risk and relapsed and refractory (RR)-MM are urgently needed to overcome drug-resistance, especially in TP53 null patients. In many cancers, nuclear exporter protein XPO1 overexpression is induced by c-Myc and/or loss of p53. Previous studies have demonstrated that many RR-MM patients show increased expression/activity of XPO1, which is associated with aberrant cytosolic localization of its protein and RNA cargoes. Selective XPO1 inhibitor Selinexor (Sel) has been approved by FDA to treat relapsed and refractory multiple myeloma (RR-MM) in combination with a bortezomib + DEX regimen. Addition of Sel to this regiment significantly improves the overall response rate (~75% vs. ~60%) and progress free survival (13.9 months vs 9.5 months), regardless of risk factors including TP53 status. However, only ~50% of RR-MM patients benefit from Sel treatment. In addition, Sel has significant dose dependent side effects. It is important to determine the mechanism of Sel-resistance in order to identify biomarker(s) to predict Sel-resistance and develop novel effective combination therapies. . In this study, we compared the response of 10 human MM cell lines to Sel treatment and identified Sel sensitive and resistant cell lines. Based on EC50, we divided the cell lines into Sel-sensitive group with EC 50 values between 80.4 nM to 211.8 nM, and Sel-resistant groups with EC 50 values &amp;gt;741.4 nM. We found that Sel sensitivity was notably independent of TP53 mutation status, as well as other genetic mutations and chromosomal translocations. In resistant cells, high dose of Sel induces an off-target Caspase-mediated apoptosis because it could not be influenced by XPO1-knockdown but could be prevented using a pan-caspase inhibitor Z-VAD. In sensitive cell lines, low dose of Sel induces a Caspase-independent type of cell death because it could not prevented by pre-treated with Z-VAD. To investigate which programmed cell death pathways Sel works through, sensitive cells were treated using with Sel + inhibitors for necroptosis, ferroptosis, pyroptosis, and parthanatos. We found only parthanatos inhibitor could significantly prevent Sel-induced cell death. The parthanatos death pathway functions via DNA-damage/poly (ADP-ribose) polymerase-1 (PARP-1) activation. While proper PARP-1 levels are required for DNA damage repair, over-activation results in the release of apoptosis-inducing factor (AIF) from mitochondria to cytoplasm which then trans-localize to the nucleus where it functions with macrophage migration inhibitory factor (MIF) to induce DNA fragmentation and cell death. We show via Western blotting and immunofluorescence imaging that upon Sel treatment, AIF shifts from the cytosol into the nucleus. Sel treatment was further assessed in combination with with Dex, where it showed a low synergistic effect with Dex in the sensitive cells but not in resistant cells, specifically when used sequentially. When assessing mechanisms of Sel resistance, phosphorylated STAT3 was uniquely observed in the resistant cell lines. To determine if STAT3 mediates resistance, resistant cell lines were pre-treated with a STAT3-PROTAC (SD36). Our study suggested that p-STAT3 predicts Sel-resistance, and Sel + STAT3 inhibitor combination is a novel effective regimen for MM, especially for these Sel-resistant cases.

Phytochemicals as Immunomodulatory Molecules in Cancer Therapeutics.

Phytochemicals are natural plant-derived products that provide significant nutrition, essential biomolecules, and flavor as part of our diet. They have long been known to confer protection against several diseases via their anti-inflammatory, immune-regulatory, anti-microbial, and several other properties. Deciphering the role of phytochemicals in the prevention, inhibition, and treatment of cancer-unrestrained cell proliferation due to the loss of tight regulation on cell growth and replication-has been the focus of recent research. Particularly, the immunomodulatory role of phytochemicals, which is pivotal in unchecked cell proliferation and metastasis, has recently been studied extensively. The immune system is a critical component of the tumor microenvironment, and it plays essential roles in both preventing and promoting oncogenesis. Immunomodulation includes stimulation, amplification, or inactivation of some stage(s) of the immune response. Phytochemicals and their products have demonstrated immune regulation, such as macrophage migration, nitric oxide synthase inhibition, lymphocyte, T-cell, and cytokine stimulation, natural killer cell augmentation, and NFκB, TNF, and apoptosis regulation. There is a dearth of extensive accounts of the immunomodulatory effects of phytochemicals in cancer; thus, we have compiled these effects with mechanistic aspects of dietary phytochemicals in cancer, highlighting promising candidates and ongoing clinical trials on immunotherapeutic strategies to mitigate oncogenesis.

Macrophage-hitchhiking interleukin-10 plasmid DNA delivery system modulates rheumatoid arthritis microenvironment via the re-polarization of macrophages

Rheumatoid arthritis (RA) is a common chronic inflammatory disease and the current treatments for RA are unsatisfactory due to the buffer barrier from the RA microenvironment. Interleukin-10 (IL-10) was found to be able to modulate rheumatoid arthritis microenvironment (RAM) by the metabolic reprogramming of macrophages. However, its clinical application has proven challenging mainly due to its short half-life, non-ideal targeting efficiency via systemic administration and poor tissue penetration. Taking advantage of the recruitment of macrophages, we constructed an innovative macrophage-hitchhiking IL-10 pDNA delivery system using the peptides bearing nuclear localization signal (NLS) and tuftsin-modified disulfide-crosslinked polymers (bPEI-SS-PEG-T) which was responsive to the intracellular glutathione concentration. Firstly, we formed NLS/DNA complexes via electrostatic interactions to enhance the nuclear entry efficiency. Then NLS/DNA complexes were combined with bPEI-SS-PEG-T to construct bPEI-SS-PEG-T/NLS/DNA NPs with sizes of 168.4 nm and zeta potentials of + 10.9 mV. As the bPEI-SS-PEG-T was biodegradable and the tuftsin peptides enhanced macrophage-mediated phagocytosis, the nanoparticles exhibited lower cytotoxicity and better cellular uptake in addition to higher nuclear entry efficiency, leading to better IL-10 plasmid transfection. In addition, these macrophage-hitchhiking nanoparticles could effectively accumulate at inflammatory sites of RA via intra-peritoneal injection due to the macrophage migration to the inflammatory joints and alleviate symptoms of inflammation in vivo by inducing the re-polarization of macrophages, which might be mediated by the inhibition of mammalian target of rapamycin (mTOR) and the induction of arginase-2 (Arg2). As a result, this macrophage-hitchhiking gene delivery system demonstrated excellent inflammation targeting ability, good nuclear entry ability, high IL-10 plasmid transfection efficiency and great therapeutic efficacy in vivo, which is promising for the treatment of rheumatoid arthritis. Besides, this delivery system also provides a novel strategy for the gene therapy and the gene delivery system design for rheumatoid arthritis and other similar inflammatory diseases from the perspective of the inflammatory microenvironment.

Protective effect of N-(E)-p-coumaroyltyrosine on LPS-induced acute inflammatory injury and signaling pathway analysis

N-trans-p-coumaroyltyrosine (N-(E)-p-coumaroyltyrosine, NPCT), extracted and purified from Abri Mollis Herba, is an amino acid amide. The defense mechanism of NPCT against inflammatory response is still unknown. In this study, lipopolysaccharide (LPS)-induced zebrafish acute inflammatory injury model was established to observe the inhibitory effect of NPCT on the aggregation of inflammatory cells in the yolk sac of zebrafish, as well as the inhibitory effect of NPCT on inflammatory and gas signaling factors. Results show that NPCT could inhibit inflammatory cell infiltration in zebrafish yolk sac, the migration and aggregation of macrophages and neutrophils to the site of inflammation, and the release of Nitric Oxide (NO) and Reactive Oxygen Species (ROS) in zebrafish, indicating that NPCT could substantially significantly prevent the development of LPS-induced acute systemic inflammation. In addition, the analysis results of RNA-seq showed that in the model group versus the administered group, the differentially expressed genes were mainly enriched to inflammatory signaling pathways, such as the NOD-like receptor signaling pathway and Toll-like receptor signaling pathway, which were down-regulated in the administered group. The TLR4, MyD88, IRAK4, NF-κB, IκB, NLRP3, Caspase-1, ASC, IL-1β, and IL-6 genes were significantly different in the transcripts, and the overall trend of the qPCR results was consistent with the transcriptome sequencing results. Therefore, NPCT had a significant inhibitory effect on LPS-induced acute inflammatory injury in zebrafish, and its anti-inflammatory mechanism may be through the regulation of key genes on the NOD-like receptor signaling pathway and Toll-like receptor signaling pathway, thereby affecting the release of relevant inflammatory cytokines.