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SUMMARY Tumor-associated macrophages (TAMs) shape tumor immunity and therapeutic efficacy. However,it is poorly understood whether and how post-translational modifications (PTMs) intrinsically affect the phenotype and function of TAMs. Here,we reveal that peptidylarginine deiminase 4 (PAD4) exhibits the highest expression among common PTM enzymes in TAMs and negatively correlates with the clinical response to immune checkpoint blockade. Genetic and pharmacological inhibition of PAD4 in macrophages prevents tumor progression in tumor-bearing mouse models,accompanied by an increase in macrophage major histocompatibility complex (MHC) class II expression and T cell effector function. Mechanistically,PAD4 citrullinates STAT1 at arginine 121,thereby promoting the interaction between STAT1 and protein inhibitor of activated STAT1 (PIAS1),and the loss of PAD4 abolishes this interaction,ablating the inhibitory role of PIAS1 in the expression of MHC class II machinery in macrophages and enhancing T cell activation. Thus,the PAD4-STAT1-PIAS1 axis is an immune restriction mechanism in macrophages and may serve as a cancer immunotherapy target. Graphical Abstract In brief Pitter et al. demonstrate that the PAD4-mediated citrullination of STAT1 in macrophages enforces the STAT1-PIAS1 interaction restraining STAT1 transcriptional activity and MHC class II machinery expression and,consequently,limits T cell-mediated anti-tumor immunity. View Publication

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent,quiescent limbal stem cells (LSCs) located at the limbus,where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood,which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study,we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover,TP63,KRT15,CXCL14,and ITGβ4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs),which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGβ4 could be used to enrich human corneal epithelial cell progenitors,which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1. View Publication

BackgroundSystemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vasculopathy and progressive fibrosis of skin and several internal organs,including lungs. Macrophages are the main cells involved in the immune-inflammatory damage of skin and lungs,and alternatively activated (M2) macrophages seem to have a profibrotic role through the release of profibrotic cytokines (IL10) and growth factors (TGFβ1). Nintedanib is a tyrosine kinase inhibitor targeting several fibrotic mediators and it is approved for the treatment of SSc-related interstitial lung disease (ILD). The study aimed to evaluate the effect of nintedanib in downregulating the profibrotic M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from SSc-ILD patients.MethodsFourteen SSc patients,fulfilling the 2013 ACR/EULAR criteria for SSc,10 SSc patients affected by ILD (SSc-ILD pts),4 SSc patients non affected by ILD (SSc pts no-ILD),and 5 voluntary healthy subjects (HSs),were recruited at the Division of Clinical Rheumatology-University of Genova,after obtaining Ethical Committee approval and patients’ informed consent. Monocytes were isolated from peripheral blood,differentiated into MDMs,and then maintained in growth medium without any treatment (untreated cells),or treated with nintedanib (0.1 and 1µM) for 3,16,and 24 h. Gene expression of macrophage scavenger receptors (CD204,CD163),mannose receptor-1 (CD206),Mer tyrosine kinase (MerTK),identifying M2 macrophages,together with TGFβ1 and IL10,were evaluated by quantitative real-time polymerase chain reaction. Protein synthesis was investigated by Western blotting and the level of active TGFβ1 was evaluated by ELISA. Statistical analysis was carried out using non-parametric Wilcoxon test.ResultsCultured untreated SSc-ILD MDMs showed a significant increased protein synthesis of CD206 (p < 0.05),CD204,and MerTK (p < 0.01),together with a significant upregulation of the gene expression of MerTK and TGFβ1 (p < 0.05; p < 0.01) compared to HS-MDMs. Moreover,the protein synthesis of CD206 and MerTK and the gene expression of TGFβ1 were significantly higher in cultured untreated MDMs from SSc-ILD pts compared to MDMs without ILD (p < 0.05; p < 0.01). In cultured SSc-ILD MDMs,nintedanib 0.1 and 1µM significantly downregulated the gene expression and protein synthesis of CD204,CD206,CD163 (p < 0.05),and MerTK (p < 0.01) compared to untreated cells after 24 h of treatment. Limited to MerTK and IL10,both nintedanib concentrations significantly downregulated their gene expression already after 16 h of treatment (p < 0.05). In cultured SSc-ILD MDMs,nintedanib 0.1 and 1µM significantly reduced the release of active TGFβ1 after 24 h of treatment (p < 0.05 vs. untreated cells).ConclusionsIn cultured MDMs from SSc-ILD pts,nintedanib seems to downregulate the profibrotic M2 phenotype through the significant reduction of gene expression and protein synthesis of M2 cell surface markers,together with the significant reduction of TGFβ1 release,and notably MerTK,a tyrosine kinase receptor involved in lung fibrosis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13075-024-03308-7. View Publication

AbstractAcute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common life‐threatening syndrome with no effective pharmacotherapy. Sepsis‐related ARDS is the main type of ARDS and is more fatal than other types. Extracellular vesicles (EVs) are considered novel mediators in the development of inflammatory diseases. Our previous research suggested that endothelial cell‐derived EVs (EC‐EVs) play a crucial role in ALI/ARDS development,but the mechanism remains largely unknown. Here,we demonstrated that the number of circulating EC‐EVs was increased in sepsis,exacerbating lung injury by targeting monocytes and reprogramming them towards proinflammatory macrophages. Bioinformatics analysis and further mechanistic studies revealed that vascular cell adhesion molecule 1 (VCAM1),overexpressed on EC‐EVs during sepsis,activated the NF‐κB pathway by interacting with integrin subunit alpha 4 (ITGA4) on the monocyte surface,rather than the tissue resident macrophage surface,thereby regulating monocyte differentiation. This effect could be attenuated by decreasing VCAM1 levels in EC‐EVs or blocking ITGA4 on monocytes. Furthermore,the number of VCAM1+ EC‐EVs was significantly increased in patients with sepsis‐related ARDS. These findings not only shed light on a previously unidentified mechanism underling sepsis‐related ALI/ARDS,but also provide potential novel targets and strategies for its precise treatment. During extra‐pulmonary sepsis,more endothelial cell‐derived extracellular vesicles (EC‐EVs) are released,which play a critical role in the development of ALI/ARDS by specifically targeting and reprogramming monocytes. VCAM1,highly expressed on these EVs,activates the NF‐κB pathway by acting on ITGA4,thus promoting the differentiation of monocytes into M1‐type macrophages. View Publication

BackgroundDifferences in male vs. female immune responses are well-documented and have significant clinical implications. While the immunomodulatory effects of sex hormones are well established,the contributions of sex chromosome complement (XX vs. XY) and gut microbiome diversity on immune sexual dimorphisms have only recently become appreciated. Here we investigate the individual and collaborative influences of sex chromosome complements and gut microbiota on humoral immune activation.MethodsMale and female Four Core Genotype (FCG) mice were immunized with heat-killed Streptococcus pneumoniae (HKSP). Humoral immune responses were assessed,and X-linked immune-related gene expression was evaluated to explain the identified XX-dependent phenotype. The functional role of Kdm6a,an X-linked epigenetic regulatory gene of interest,was evaluated ex vivo using mitogen stimulation of B cells. Additional influences of the gut microbiome on sex chromosome-dependent B cell activation was also evaluated by antibiotically depleting gut microbiota prior to HKSP immunization. Reconstitution of the depleted microbiome with short-chain fatty acid (SCFA)-producing bacteria tested the impact of SCFAs on XX-dependent immune activation.ResultsXX mice exhibited higher HKSP-specific IgM-secreting B cells and plasma cell frequencies than XY mice,regardless of gonadal sex. Although Kdm6a was identified as an X-linked gene overexpressed in XX B cells,inhibition of its enzymatic activity did not affect mitogen-induced plasma cell differentiation or antibody production in a sex chromosome-dependent manner ex vivo. Enhanced humoral responses in XX vs. XY immunized FCG mice were eliminated after microbiome depletion,indicating that the microbiome contributes to the identified XX-dependent immune enhancement. Reconstituting microbiota-depleted mice with select SCFA-producing bacteria enhanced fecal SCFA concentrations and increased humoral responses in XX,but not XY,FCG mice. However,exposure to the SCFA propionate alone did not enhance mitogenic B cell stimulation in ex vivo studies.ConclusionsFCG mice have been used to assess sex hormone and sex chromosome complement influences on various sexually dimorphic traits. The current study indicates that the gut microbiome impacts humoral responses in an XX-dependent manner,suggesting that the collaborative influence of gut bacteria and other sex-specific factors should be considered when interpreting data aimed at delineating the mechanisms that promote sexual dimorphism.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13293-024-00597-0. Highlights Humoral immune responses against HKSP immunization are influenced by the possession of an XX vs. XY sex chromosome complement. While gonadal sex differentially influenced the number of antigen-specific IgM-secreting cells,the overall percentage of CD138 + plasma cells generated in response to HKSP immunization was not influenced by gonadal sex.Kdm6a is overexpressed in XX vs. XY B cells and splenocytes of HKSP-immunized mice and is demonstrated to be biallelically expressed in a subset of B cells.Ex vivo inhibition of KDM6a enzymatic activity promotes plasma cell differentiation in response to mitogenic stimulation. However,this effect was not sex chromosome-dependent. KDM6a inhibition did not impact total IgM concentrations in culture supernatants following mitogenic stimulation.XX-dependent immune enhancement is microbiome-dependent. Reconstitution of the antibiotic-depleted gut microbiome with select SCFA-producing bacteria rescued the XX-dependent immune phenotype observed in XX,but not XY,FCG mice. Supplementary InformationThe online version contains supplementary material available at 10.1186/s13293-024-00597-0. Plain language summaryMale and female immune systems differ in their ability to respond to infectious challenge. While males tend to be more susceptible to infection and produce lower amounts of antibodies in response to vaccination,females are more prone to develop autoimmune and inflammatory diseases. Key contributors to these differences include sex hormones,sex chromosome complement (XX in females vs. XY in males),and distinct gut microbial communities capable of regulating immune activation. While each factor has been studied individually,this research underscores the potential for these factors to collaboratively impact immune activation. Here,possession of an XX vs. XY sex chromosome complement was demonstrated to enhance antibody responses to heat-killed Streptococcus pneumoniae vaccination. While attempting to determine the underlying cause of this immune enhancement,the gut microbiome was identified to play a critical role. In the absence of an intact gut microbiome,XX immune activation was reduced to levels similar to those seen in XY sex chromosome complement-possessing mice. Replacement of the depleted gut microbiomes with select SCFA-producing bacterial species enhanced SCFA levels in antibiotic-treated mice and rescued the XX-dependent immune enhancement,suggesting a SCFA-mediated contribution. Further studies are needed to determine exactly how these select bacteria impact immune activation in a sex chromosome complement-dependent manner. Our findings highlight the need to consider the collaborative effects of individual sex-specific factors when attempting to understand immune sex biases,as a better understanding of these interactions will likely pave the way for improving therapeutics and vaccines tailored to both sexes.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13293-024-00597-0. View Publication

AbstractThe effect of targeted therapeutics on anticancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2,and the therapeutic effect has been partially attributed to GCN2 activation. Because ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function,we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T-cell activity,which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration,and attenuated transcriptional programs required for PMN development. Moreover,we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways,and increased activity of transcriptional regulons driven by Atf5,Mafg,and Zbtb7a. This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest,skewing immature MDSC development toward monocytic lineage cells. In vivo,we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally,in the tumor immune infiltrate. Thus,our data reveal transcriptional networks that govern MDSC developmental programs,and the impact of GCN2 stress signaling on the innate immune landscape in tumors,providing novel insight into potentially beneficial off-target effects of dabrafenib.Significance:An important,but poorly understood,aspect of targeted therapeutics for cancer is the effect on antitumor immune responses. This article shows that off-target effects of dabrafenib activating the kinase GCN2 impact MDSC development and function reducing PMN-MDSCs in vitro and in vivo. This has important implications for our understanding of how this BRAF inhibitor impacts tumor growth and provides novel therapeutic target and combination possibilities. View Publication

Understanding the molecular and cellular processes involved in lung epithelial regeneration may fuel the development of therapeutic approaches for lung diseases. We combine mouse models allowing diphtheria toxin-mediated damage of specific epithelial cell types and parallel GFP-labeling of functionally dividing cells with single-cell transcriptomics to characterize the regeneration of the distal lung. We uncover cell types,including Krt13+ basal and Krt15+ club cells,detect an intermediate cell state between basal and goblet cells,reveal goblet cells as actively dividing progenitor cells,and provide evidence that adventitial fibroblasts act as supporting cells in epithelial regeneration. We also show that diphtheria toxin-expressing cells can persist in the lung,express specific inflammatory factors,and transcriptionally resemble a previously undescribed population in the lungs of COVID-19 patients. Our study provides a comprehensive single-cell atlas of the distal lung that characterizes early transcriptional and cellular responses to concise epithelial injury,encompassing proliferation,differentiation,and cell-to-cell interactions. This study uses single-cell transcriptomics to examine how lung cells respond to targeted damage. The authors employ genetically modified mouse models and cell sorting to enrich for rare,actively dividing cells,revealing cell types/states and alternative differentiation paths. View Publication

IntroductionLoss of proteasome function,proteinopathy,and proteotoxicity may cause neurodegeneration across the human lifespan in several forms of brain injury and disease. Drugs that activate brain proteasomes in vivo could thus have a broad therapeutic impact in neurology.MethodsUsing pigs,a clinically relevant large animal with a functionally compartmental gyrencephalic cerebral cortex,we evaluated the localization and biochemical activity of brain proteasomes and tested the ability of small molecules to activate brain proteasomes.ResultsBy Western blotting,proteasome protein subunit PSMB5 and PSMA3 levels were similar in different pig brain regions. Immunohistochemistry for PSMB5 showed localization in the cytoplasm (diffuse and particulate) and nucleus (cytoplasm < nucleus). Some PSMB5 immunoreactivity was colocalized with mitochondrial (voltage-gated anion channel and cyclophilin D) and cell death (Aven) proteins in the neuronal soma and neuropil in the neocortex of pig and human brains. In the nucleus,PSMB5 immunoreactivity was diffuse,particulate,and clustered,including perinucleolar decorations. By fluorogenic assay,proteasome chymotrypsin-like activities (CTL) in crude tissue soluble fractions were generally similar within eight different pig brain regions. Proteasome CTL activity in the hippocampus was correlated with activity in nasal mucosa biopsies. In pilot analyses of subcellular fractions of pig cerebral cortex,proteasome CTL activity was highest in the cytosol and then ~50% lower in nuclear fractions; ~15–20% of total CTL activity was in pure mitochondrial fractions. With in-gel activity assay,26S-singly and -doubly capped proteasomes were the dominant forms in the pig cerebral cortex. With a novel in situ histochemical activity assay,MG132-inhibitable proteasome CTL activity was localized to the neuropil,as a mosaic,and to cell bodies,nuclei,and centrosome-like perinuclear satellites. In piglets treated intravenously with pyrazolone derivative and chlorpromazine over 24 h,brain proteasome CTL activity was modestly increased.DiscussionThis study shows that the proteasome in the pig brain has relative regional uniformity,prominent nuclear and perinuclear presence with catalytic activity,a mitochondrial association with activity,26S-single cap dominance,and indications from small molecule systemic administration of pyrazolone derivative and chlorpromazine that brain proteasome function appears safely activable. View Publication

SUMMARYThe mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-weeks supplementation of antiretroviral therapy (ART) with metformin,an indirect mTOR inhibitor used in type-2 diabetes treatment,reduced mTOR activation and HIV transcription in colon-infiltrating CD4+ T-cells,together with systemic inflammation in nondiabetic people with HIV-1 (PWH). Herein,we investigated the antiviral mechanisms of metformin. In a viral outgrowth assay performed with CD4+ T-cells from ART-treated PWH,and upon infection in vitro with replication-competent and VSV-G-pseudotyped HIV-1,metformin decreased virion release,but increased the frequency of productively infected CD4lowHIV-p24+ T-cells. These observations coincided with increased BST2/Tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression,and improved recognition of productively infected T-cells by HIV-1 Envelope antibodies. Thus,metformin exerts pleiotropic effects on post-transcription/translation steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH. Graphical Abstract View Publication

BackgroundDendritic cell (DC)-mediated antigen presentation is essential for the priming and activation of tumor-specific T cells. However,few drugs that specifically manipulate DC functions are available. The identification of drugs targeting DC holds great promise for cancer immunotherapy.MethodsWe observed that type 1 conventional DCs (cDC1s) initiated a distinct transcriptional program during antigen presentation. We used a network-based approach to screen for cDC1-targeting therapeutics. The antitumor potency and underlying mechanisms of the candidate drug were investigated in vitro and in vivo.ResultsSitagliptin,an oral gliptin widely used for type 2 diabetes,was identified as a drug that targets DCs. In mouse models,sitagliptin inhibited tumor growth by enhancing cDC1-mediated antigen presentation,leading to better T-cell activation. Mechanistically,inhibition of dipeptidyl peptidase 4 (DPP4) by sitagliptin prevented the truncation and degradation of chemokines/cytokines that are important for DC activation. Sitagliptin enhanced cancer immunotherapy by facilitating the priming of antigen-specific T cells by DCs. In humans,the use of sitagliptin correlated with a lower risk of tumor recurrence in patients with colorectal cancer undergoing curative surgery.ConclusionsOur findings indicate that sitagliptin-mediated DPP4 inhibition promotes antitumor immune response by augmenting cDC1 functions. These data suggest that sitagliptin can be repurposed as an antitumor drug targeting DC,which provides a potential strategy for cancer immunotherapy. View Publication

AbstractT cells in the human female genital tract (FGT) are key mediators of susceptibility to and protection from infection,including HIV and other sexually transmitted infections. There is a critical need for increased understanding of the distribution and activation of T cell populations in the FGT,but current sampling methods require a healthcare provider and are expensive,limiting the ability to study these populations longitudinally. To address these challenges,we have developed a method to sample immune cells from the FGT utilizing disposable menstrual discs which are noninvasive,self-applied,and low in cost. To demonstrate reproducibility,we sampled the cervicovaginal fluid of healthy,reproductive-aged individuals using menstrual discs across 3 sequential days. Cervicovaginal fluid was processed for cervicovaginal cells,and high-parameter flow cytometry was used to characterize immune populations. We identified large numbers of live,CD45+ leukocytes,as well as distinct populations of T cells and B cells. Within the T cell compartment,activation and suppression status of T cell subsets were consistent with previous studies of the FGT utilizing current approaches,including identification of both tissue-resident and migratory populations. In addition,the T cell population structure was highly conserved across days within individuals but divergent across individuals. Our approach to sample immune cells in the FGT with menstrual discs will decrease barriers to participation and empower longitudinal sampling in future research studies. View Publication

BackgroundStreptococcus pyogenes (group A streptococcus,GAS) causes a variety of diseases ranging from mild superficial infections of the throat and skin to severe invasive infections,such as necrotizing soft tissue infections (NSTIs). Tissue passage of GAS often results in mutations within the genes encoding for control of virulence (Cov)R/S two component system leading to a hyper-virulent phenotype. Dendritic cells (DCs) are innate immune sentinels specialized in antigen uptake and subsequent T cell priming. This study aimed to analyze cytokine release by DCs and other cells of monocytic origin in response to wild-type and natural covR/S mutant infections.MethodsHuman primary monocyte-derived (mo)DCs were used. DC maturation and release of pro-inflammatory cytokines in response to infections with wild-type and covR/S mutants were assessed via flow cytometry. Global proteome changes were assessed via mass spectrometry. As a proof-of-principle,cytokine release by human primary monocytes and macrophages was determined.ResultsIn vitro infections of moDCs and other monocytic cells with natural GAS covR/S mutants resulted in reduced secretion of IL-8 and IL-18 as compared to wild-type infections. In contrast,moDC maturation remained unaffected. Inhibition of caspase-8 restored secretion of both molecules. Knock-out of streptolysin O in GAS strain with unaffected CovR/S even further elevated the IL-18 secretion by moDCs. Of 67 fully sequenced NSTI GAS isolates,28 harbored mutations resulting in dysfunctional CovR/S. However,analyses of plasma IL-8 and IL-18 levels did not correlate with presence or absence of such mutations.ConclusionsOur data demonstrate that strains,which harbor covR/S mutations,interfere with IL-18 and IL-8 responses in monocytic cells by utilizing the caspase-8 axis. Future experiments aim to identify the underlying mechanism and consequences for NSTI patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12929-024-01014-9. View Publication