Decreased IL-10 Production Research Articles

Inhibition of BCAT1-mediated cytosolic leucine metabolism regulates Th17 responses via the mTORC1-HIF1α pathway

Branched-chain amino acids (BCAAs), particularly leucine, are indispensable AAs for immune regulation through metabolic rewiring. However, the molecular mechanism underlying this phenomenon remains unclear. Our investigation revealed that T-cell receptor (TCR)-activated human CD4+ T cells increase the expression of BCAT1, a cytosolic enzyme responsible for BCAA catabolism, and SLC7A5, a major BCAA transporter. This upregulation facilitates increased leucine influx and catabolism, which are particularly crucial for Th17 responses. Activated CD4+ T cells induce an alternative pathway of cytosolic leucine catabolism, generating a pivotal metabolite, β-hydroxy β-methylbutyric acid (HMB), by acting on BCAT1 and 4-hydroxyphenylpyruvate dioxygenase (HPD)/HPD-like protein (HPDL). Inhibition of BCAT1-mediated cytosolic leucine metabolism, either with BCAT1 inhibitor 2 (Bi2) or through BCAT1, HPD, or HPDL silencing using shRNA, attenuates IL-17 production, whereas HMB supplementation abrogates this effect. Mechanistically, HMB contributes to the regulation of the mTORC1-HIF1α pathway, a major signaling pathway for IL-17 production, by increasing the mRNA expression of HIF1α. This finding was corroborated by the observation that treatment with L-β-homoleucine (LβhL), a leucine analog and competitive inhibitor of BCAT1, decreased IL-17 production by TCR-activated CD4+ T cells. In an in vivo experimental autoimmune encephalomyelitis (EAE) model, blockade of BCAT1-mediated leucine catabolism, either through a BCAT1 inhibitor or LβhL treatment, mitigated EAE severity by decreasing HIF1α expression and IL-17 production in spinal cord mononuclear cells. Our findings elucidate the role of BCAT1-mediated cytoplasmic leucine catabolism in modulating IL-17 production via HMB-mediated regulation of mTORC1-HIF1α, providing insights into its relevance to inflammatory conditions.

Thirty days of double-strain probiotic supplementation increases monocyte phagocytosis in marathon runners.

Marathon runners, subjected to intense training regimens and prolonged, exhaustive exercises, often experience a compromised immune response. Probiotic supplementation has emerged as a potential remedy to mitigate the impact of prolonged exercise on athletes. Consequently, this study sought to assess the influence of probiotic supplementation on monocyte functionality both before and after the official marathon race. Twenty-seven runners were randomly and double-blindly assigned to two groups: placebo (n 13) and probiotic (PRO) (n 14). Over 30 d, both groups received supplements - placebo sachets containing maltodextrin (5 g/d) and PRO sachets containing 1 × 1010 colony-forming unit Lactobacillus acidophilus and 1 × 1010 colony-forming unit Bifidobacterium bifidum subsp. lactis. Blood samples were collected, and immunological assays, including phagocytosis, hydrogen peroxide production, cytokine levels and monocyte immunophenotyping, were conducted at four different intervals: baseline (start of supplementation/30 d pre-marathon), 24 h-before (1 d pre-marathon), 1 h-after (1 h post-marathon) and 5 d-after (5 d post-marathon). Monocyte populations remained consistent throughout the study. A notable increase in phagocytosis was observed in the PRO group after 30 d of supplementation. Upon lipopolysaccharide stimulation, both PRO and placebo groups exhibited decreased IL-8 production. However, after the marathon race, IL-15 stimulation demonstrated increased levels of 5 d-after, while IL-1-β, IL-8, IL-10, IL-15 and TNF-α varied across different intervals, specifically within the PRO group. Probiotic supplementation notably enhanced the phagocytic capacity of monocytes. However, these effects were not sustained post-marathon.

Accumulation of Lipid Droplets Induced by Listeria monocytogenes in Macrophages: Implications for Survival and Evasion of Innate Immunity.

Listeriosis, caused by Listeria monocytogenes (L.m.), poses a significant public health concern as one of the most severe foodborne diseases. The pathogenesis of L.m. involves critical steps such as phagosome rupture and escape upon internalization. Throughout infection, L.m. influences various host processes, including lipid metabolism pathways, yet the role of lipid droplets (LDs) remains unclear. Here, we reported a rapid, time-dependent increase in LD formation in macrophages induced by L.m. LD biogenesis was found to be dependent on L.m. viability and virulence genes, particularly on the activity of the pore-forming protein listeriolysin O (LLO). The prevention of LD formation by inhibiting diacylglycerol O-acyltransferase 1 (DGAT1) and cytosolic phospholipase A2 (cPLA2) significantly reduced intracellular bacterial survival, impaired prostaglandin E2 (PGE2) synthesis, and decreased IL-10 production. Additionally, inhibiting LD formation led to increased levels of TNF-α and IFN-β. Collectively, our data suggest a role for LDs in promoting L.m. cell survival and evasion within macrophages.

Septin-dependent defense mechanisms against Pseudomonas aeruginosa are stalled in cystic fibrosis bronchial epithelial cells

Airway epithelial cells form a physical barrier against inhaled pathogens and coordinate innate immune responses in the lungs. Bronchial cells in people with cystic fibrosis (pwCF) are colonized by Pseudomonas aeruginosa because of the accumulation of mucus in the lower airways and an altered immune response. This leads to chronic inflammation, lung tissue damage, and accelerated decline in lung function. Thus, identifying the molecular factors involved in the host response in the airways is crucial for developing new therapeutic strategies. The septin (SEPT) cytoskeleton is involved in tissue barrier integrity and anti-infective responses. SEPT7 is critical for maintaining SEPT complexes and for sensing pathogenic microbes. In the lungs, SEPT7 may be involved in the epithelial barrier resistance to infection; however, its role in cystic fibrosis (CF) P. aeruginosa infection is unknown. This study aimed to investigate the role of SEPT7 in controlling P. aeruginosa infection in bronchial epithelial cells, particularly in CF. The study findings showed that SEPT7 encages P. aeruginosa in bronchial epithelial cells and its inhibition downregulates the expression of other SEPTs. In addition, P. aeruginosa does not regulate SEPT7 expression. Finally, we found that inhibiting SEPT7 expression in bronchial epithelial cells (BEAS-2B 16HBE14o- and primary cells) resulted in higher levels of internalized P. aeruginosa and decreased IL-6 production during infection, suggesting a crucial role of SEPT7 in the host response against this bacterium. However, these effects were not observed in the CF cells (16HBE14o-/F508del and primary cells) which may explain the persistence of infection in pwCF. The study findings suggest the modification of SEPT7 expression as a potential approach for the anti-infective control of P. aeruginosa, particularly in CF.

STAT3 promotes cytoplasmic-nuclear translocation of RNA-binding protein HuR to inhibit IL-1β-induced IL-8 production

Signal transducer and activator of transcription 3 (STAT3) functions to regulate inflammation and immune response, but its mechanism is not fully understood. We report here that STAT3 inhibitors Stattic and Niclosamide up-regulated IL-1β-induced IL-8 production in C33A, CaSki, and Siha cervical cancer cells. As expected, IL-1β-induced IL-8 production was also up-regulated through the molecular inhibition of STAT3 by use of CRISPR/Cas9 technology. Unexpectedly, IL-1β induced IL-8 production via activating ERK and P38 signal pathways, but neither STAT3 inhibitors nor STAT3 knockout affected IL-1β-induced signal transduction, suggesting that STAT3 decreases IL-8 production not via inhibition of signal transduction. To our surprise, STAT3 inhibition increased the stabilization, and decreased the degradation of IL-8 mRNA, suggesting a post-transcriptional regulation of IL-1β-induced IL-8. Moreover, Dihydrotanshinone I, an inhibitor of RNA-binding protein HuR, down-regulated IL-1β-induced IL-8 dose-dependently. HuR inhibition by CRISPR/Cas9 also decreased IL-8 production induced by IL-1β. Mechanistically, co-immunoprecipitation results showed that STAT3 did not react with HuR directly, but STAT3 inhibition increased the protein levels of HuR in cytoplasm. And IL-6 activation of STAT3 induced HuR cytoplasmic-nuclear transport. Taken together, these results suggest that STAT3 contributes to HuR nuclear localization and inhibits Il-1β-induced IL-8 production through this non-transcriptional mechanism.

Leukocyte dysfunction and reduced CTLA-4 expression are associated with perianal Crohn's disease.

Although perianal Crohn's disease (PCD) is highly associated with the exacerbated inflammation, the molecular basis and immunological signature that distinguish patients who present a history of perianal lesions are still unclear. This paper aims to define immunological characteristics related to PCD. In this cross-sectional observational study, we enrolled 20 healthy controls and 39 CD patients. Blood samples were obtained for the detection of plasma cytokines and lipopolysaccharides (LPS). Peripheral blood mononuclear cells (PBMCs) were phenotyped by flow cytometry. Leukocytes were stimulated with LPS or anti-CD3/anti-CD28 antibodies. Our results show that CD patients had augmented plasma interleukin (IL)-6 and LPS. However, their PBMC was characterized by decreased IL-6 production, while patients with a history of PCD produced higher IL-6, IL-8, and interferon-γ, along with decreased tumor necrosis factor alpha (TNF). CD patients had augmented FoxP3 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) regulatory markers, though the PCD subjects presented a significant reduction in CTLA-4 expression. CTLA-4 as well as IL-6 and TNF responses were able to distinguish the PCD patients from those who did not present perianal complications. In conclusion, IL-6, TNF, and CTLA-4 exhibit a distinct expression pattern in CD patients with a history of PCD, regardless of disease activity. These findings clarify some mechanisms involved in the development of the perianal manifestations and may have a great impact on the disease management.

Cannabidiol Mediates In Vitro Attenuation of Proinflammatory Cytokine Responses in Psoriatic Disease.

Background: Cannabidiol (CBD), a substance that belongs to the phytocannabinoids, appears to exert antioxidant, neuroprotective, antipsychotic, anticonvulsant, and anticancer properties. Recent evidence supports the immunoregulatory effect of CBD on autoimmune and/or inflammatory disease. Psoriasis is a chronic skin disease. The main immune cell population involved in the pathogenesis of the disease is the interleukin- (IL-) 17-producing T helper (Th) 17 subset. Other subpopulations, such as interferon-γ (IFNγ) -producing Th1 and T cytotoxic (Tc) 1, IL-17-producing Tc17, as well as natural killer (NK) and natural killer T cells (NKT) have been implicated in psoriasis development. Purpose: The aim of the present study was to evaluate the in vitro effect of CBD on the aforementioned subpopulations isolated from patients with psoriasis using flow cytometry. Methods: Cells were stimulated in the presence or absence of CBD, stained and examined using surface and intracellular markers. Results: CBD decreased IL-17 production within the CD3, Th, and NKT cell compartments and IFNγ production within the CD3 compartment in cells isolated from patients with psoriasis. Interestingly, CBD supplementation did not inhibit production of proinflammatory cytokines in cells isolated from healthy individuals. On the contrary, IFNγ-producing Th, Tc, and NK cells increased after CBD supplementation. Conclusion: CBD provides anti-inflammatory effects in T cells isolated from patients with psoriasis. Our results could be the impetus for future investigations regarding the immunomodulatory properties of CBD and its utilization for development of CBD-containing antipsoriatic agents.

The use of xenogenic testicular antigens for induction of antitumor reactions

Testicular antigens (TAGs) are normally expressed only by cells of testicular and placental tissues. Human immune system is tolerant to TAG, but if the integrity of the testicular membranes is disrupted, these antigens, entering the bloodstream, induce autoimmune reactions for eliminating them from the body. In malignancy, TAGs begin to be expressed by tumor cells of the liver, breast, pancreas, intestine, and lung. Immunological recognition of these AGs leads to autoimmune reactions against these AGs, i.e. antitumor reactions in the body. We used xenogenic TAGs derived from ram testis to increase TAG immunogenicity. The use of ram TAGs is justified by the fact that TAGs are evolutionarily conserved molecules and there is a high degree of homology between human and animal TAGs.The purpose of the study was to evaluate the lifespan of tumor-bearing mice and parameters of cellular immunity in various options for immunizing mice with ram TAGs.Material and Methods. C57BL/6 mice were used. The efficacy of therapeutic or prophylactic vaccination with xenogenic TAGs was studied by changing lifespan of B16 and LLC tumor-bearing mice. Formation of immune responses was evaluated by proliferative ability of splenocytes to respond to vaccination and control AGs and by their production of IFN-gamma and IL-10.Results. In the LLC carcinoma model with a preventive vaccination option, the lifespan of mice with syngeneic vaccination did not differ from the tumor control; the lifespan of mice with xenogeneic vaccination increased by 60%. In therapeutic vaccination option, no significant differences in lifespan of vaccinated mice were found. A significant increase in the proliferative activity of splenocytes in response to tumor AGs was found in both LLC- and B16 tumor-bearing mice previously vaccinated with xenogenic TAGs. The increased IFN-gamma production by splenocytes was observed in B16 and LLC tumorbearing mice with xenogeneic vaccination. The IFN-gamma production by splenocytes in tumor-bearing mice with syngeneic vaccination was not increased. A significant decrease in IL-10 production was noted in mice with xenogeneic vaccination.

The NLRP3 molecule influences the therapeutic effects of mesenchymal stem cells through Glut1-mediated energy metabolic reprogramming

IntroductionNumerous studies demonstrated that NLRP3 has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Mesenchymal stem cells (MSCs) regulated the NLRP3 inflammasome, which has emerged as a novel therapeutic approach for treating IBD. ObjectivesThe exact role of NLRP3 in regulating MSCs’ function is unclear. Our study aimed to explore how NLRP3 affects the therapeutic effects of MSCs in colitis. MethodsWe extracted MSCs from the bone marrow of C57BL/6 mice and Nlrp3 KO mice, and identified them using differentiation assays and flow cytometry. In vitro, Both WT MSCs and Nlrp3 KO MSCs were stimulated with inflammatory factor Lipopolysaccharide (LPS), and only WT MSCs were stimulated with varying concentrations of the NLRP3 inhibitor MCC950, then, quantified IL-10 levels in the supernatant. RNA-seq was performed to examine gene expression patterns and Seahorse was used to assess oxidative phosphorylation (OXPHOS) and glycolysis levels. Western blot was used to evaluate protein expression. In vivo, we treated DSS-induced colitis with either WT or Nlrp3 KO MSCs, monitoring weight, measuring colon length, and further evaluation. We also treated DSS-induced colitis with pretreated MSCs (BAY876, oe-Glut1, or oe-NLRP3), following the same experimental procedures as described above. ResultsOur results demonstrate that Nlrp3 deletion did not affect MSC phenotypes, but rather promoted osteogenic differentiation. However, the absence of Nlrp3 reduced IL-10 production in MSCs in the presence of LPS, leading to impaired protection on DSS-induced colitis. Conversely, overexpression of NLRP3 promotes the production of IL-10, enhancing therapeutic effects. Further investigation revealed that Nlrp3 deficiency downregulated Glut1 expression and glycolysis activation in MSCs, resulting in decreased IL-10 production. Notably, overexpressing Glut1 in Nlrp3 KO MSCs restored their therapeutic effect that was previously dampened due to Nlrp3 deletion. ConclusionOur findings demonstrate that NLRP3 heightens the therapeutic effects of MSC treatment on DSS-induced colitis.

Streptococcal Arginine Deiminase Inhibits T Lymphocyte Differentiation In Vitro.

Pathogenic microbes use arginine-metabolizing enzymes as an immune evasion strategy. In this study, the impact of streptococcal arginine deiminase (ADI) on the human peripheral blood T lymphocytes function in vitro was studied. The comparison of the effects of parental strain (Streptococcus pyogenes M49-16) with wild type of ArcA gene and its isogenic mutant with inactivated ArcA gene (Streptococcus pyogenes M49-16delArcA) was carried out. It was found that ADI in parental strain SDSC composition resulted in a fivefold decrease in the arginine concentration in human peripheral blood mononuclear cell (PBMC) supernatants. Only parental strain SDSCs suppressed anti-CD2/CD3/CD28-bead-stimulated mitochondrial dehydrogenase activity and caused a twofold decrease in IL-2 production in PBMC. Flow cytometry analysis revealed that ADI decreased the percentage of CM (central memory) and increased the proportion of TEMRA (terminally differentiated effector memory) of CD4+ and CD8+ T cells subsets. Enzyme activity inhibited the proliferation of all CD8+ T cell subsets as well as CM, EM (effector memory), and TEMRA CD4+ T cells. One of the prominent ADI effects was the inhibition of autophagy processes in CD8+ CM and EM as well as CD4+ CM, EM, and TEMRA T cell subsets. The data obtained confirm arginine's crucial role in controlling immune reactions and suggest that streptococcal ADI may downregulate adaptive immunity and immunological memory.

PARP2 downregulation in T cells ameliorates lipopolysaccharide-induced inflammation of the large intestine.

T cell-dependent inflammatory response with the upregulation of helper 17 T cells (Th17) and the downregulation of regulatory T cells (Treg) accompanied by the increased production of tumor necrosis alpha (TNFa) is characteristic of inflammatory bowel diseases (IBD). Modulation of T cell response may alleviate the inflammation thus reduce intestinal damage. Poly(ADP-ribose) polymerase-2 (PARP2) plays role in the development, differentiation and reactivity of T cell subpopulations. Our aim was to investigate the potential beneficial effect of T cell-specific PARP2 downregulation in the lipopolysaccharide (LPS) induced inflammatory response of the cecum and the colon. Low-dose LPS was injected intraperitoneally to induce local inflammatory response, characterized by increased TNFa production, in control (CD4Cre; PARP2+/+) and T cell-specific conditional PARP2 knockout (CD4Cre; PARP2f/f) mice. TNFa, IL-1b, IL-17 levels were measured by ELISA, oxidative-nitrative stress was estimated by immunohistochemistry, while PARP1 activity, p38 MAPK and ERK phosphorylation, and NF-kB expression in large intestine tissue samples were examined by Western-blot. Systemic & local T cell subpopulation; Th17 and Treg alterations were also investigated using flowcytometry and immunohistochemistry. In control animals, LPS induced intestinal inflammation with increased TNFa production, while no significant elevation of TNFa production was observed in T cell-specific PARP2 knockout animals. The absence of LPS-induced elevation in TNFa levels was accompanied by the absence of IL-1b elevation and the suppression of IL-17 production, showing markedly reduced inflammatory response. The increase in oxidative-nitrative stress and PARP1-activation was also absent in these tissues together with altered ERK and NF-kB activation. An increase in the number of the anti-inflammatory Treg cells in the intestinal mucosa was observed in these animals, together with the reduction of Treg count in the peripheral circulation. Our results confirmed that T cell-specific PARP2 downregulation ameliorated LPS-induced colitis. The dampened TNFa production, decreased IL-17 production and the increased intestinal regulatory T cell number after LPS treatment may be also beneficial during inflammatory processes seen in IBD. By reducing oxidative-nitrative stress and PARP1 activation, T cell-specific PARP2 downregulation may also alleviate intestinal tissue damage.

TLR3 recognition of viral double-stranded RNA in human dental pulp cells is important for the innate immunity

Dental caries or trauma can expose human dental pulp cells (DPCs) to various oral microorganisms, which play an important role in the development of an innate immune response. In the present study, we examined the expression of Toll-like receptors (TLRs) for sensing microbe-associated molecular patterns in human DPCs. Interestingly, real-time PCR analysis demonstrated that TLR3 is the most highly expressed among 10 different TLRs in human DPCs. Poly(I:C), a representative TLR3 ligand mimicking viral double-stranded RNA, potently induced IL-8 expression in a time- and dose-dependent manner. Concordantly, poly(I:C) treatment substantially increased the expression of pro-inflammatory cytokines and chemokines such as IL-6, CCL2, and CXCL10. Human DPCs transfected with TLR3 siRNA exhibited decreased IL-8 production compared with non-targeting siRNA-transfected cells, suggesting that the expression of poly(I:C)-induced inflammatory cytokines is dependent on TLR3. IL-8 secretion induced by poly(I:C) was down-regulated by MAP kinase inhibitors, indicating that the MAP kinase pathway contributes to IL-8 production. Furthermore, C/EBPβ and NF-κB were essential transcriptional factors for poly(I:C)-induced IL-8 expression, as demonstrated by the transient transfection and reporter gene assay. Since lipoproteins are known as major immunostimulatory components of bacteria, human DPCs were treated with poly(I:C) together with Pam2CSK4, a synthetic lipopeptide mimicking bacterial lipoproteins. Pam2CSK4 and poly(I:C) co-treatment synergistically increased IL-8 production in comparison to Pam2CSK4 or poly(I:C) alone, implying that co-infection of viruses and bacteria can synergistically induce inflammatory responses in the dental pulp. Taken together, these results suggest that human DPCs potentially sense and respond to viral double-stranded RNAs, leading to effective induction of innate immune responses.

Unveiling the molecular basis of inflamm-aging induced by advanced glycation end products (AGEs)-modified human serum albumin (AGE-HSA) in patients with different immune-mediated diseases

Increased serum advanced glycation end products (AGEs) are commonly found in the patients with Diabetes mellitus (DM), aging-related diseases, and immune-mediated diseases. These diseases are notorious for vasculopathy, immune dysfunctions, and low-grade inflammation mimicking inflamm-aging. However, the molecular basis of inflamm-aging related to AGEs remains elucidation. In this study, we incubated human serum albumin (HSA) and glucose at 37 °C in 5% CO2 incubator for 0–180 days to generate AGE-HSA. We found the mixture gradually changing the color from transparancy to brown color and increased molecular weight during incubation. The pH value also gradually decreased from 7.2 to 5.4 irrelevant to ionic charge or [Ca2+] concentration, but dependent on gradual glycation of the alkaline amino acids, lysine and arginine. Functionally, 40 μg/mL of AGE-HSA decreased IL-2 production from human Jurkat T cell line via suppressing p-STAT3, p-STAT4, and p-STAT6 with an increased tendency of senescence-associated β-galactosidase (SA-βgal) expression but irrelevant to change of Th1/Th2/Treg subpopulations. In contrast, AGE-HSA enhanced CC motif chemokine ligand 5 (CCL-5), IL-8, macrophage migration inhibitor factor (MIF), and interleukin 1 receptor antagonist (IL-1Ra) but suppressed SA-βgal expression by human macrophage-like THP-1 cells. Interestingly, AGE-HSA abrogated the HSA-induced soluble intercellular adhesion molecules 1 (sICAM-1), sE-selectin and endothelin release from human coronary artery endothelial cells (HCAEC) and enhanced SA-βgal expression. The accelerated and increased HSA glycations by individual inflammation-related cytokine such as IL-2, IL-6, IL-17, TGF-β, or TNF-α in the in vitro study reflect increased serum AGE levels in patients with immune-mediated diseases. In conclusion, AGE-HSA can exert immunosuppresive, inflammatory and vasculopathic effects mimicking inflamm-aging in these patients.

Histone Demethylase JARID1C/KDM5C Regulates Th17 T Cells in Diabetic Wounds by Increasing IL-6 Expression in Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDCs) are first responders to tissue damage and interact with wound CD4 T cells to orchestrate tissue repair. In diabetes, the role of pDCs and their subsequent influence on wound CD4+ T cells and inflammation during tissue repair is unknown. Using human diabetic wound single cell sequencing, we identified that human wound CD4+ T-cells are primarily of the inflammatory TH17 phenotype. Thus, we hypothesized that pDCs in the wounds may regulate this T-cell phenotype, contributing to chronic inflammation. We utilized human diabetic wound single cell sequencing and analyzed murine wounds from diabetic and nondiabetic mice. These tissue were subjected to pDC isolation by magnetic sorting, flow cytometry, chromatin immunoprecipitation, quantitative polymerase chain reaction, histone arrays, and enzyme-linked immunosorbent assays. We found that diabetic pDC produced significantly more IL-6 compared to controls. This is important as IL-6 has been shown to skew CD4+ T cells toward a TH-17 phenotype in tissues. We identified that histone demethylase JARID1C was significantly decreased in diabetic pDCs compared to controls. JARID1C is an epigenetic enzyme responsible for demethylating lysine 4 on histone 3, resulting in gene transcription inhibition. We demonstrate that type 1 interferons regulate JARID1C expression via TYK2/JAK1,3 mechanism. Finally, we show that in vitro administration of recombinant JARID1C decreases IL-6 production and TH17 T-cell expression. Taken together, these data suggest that, in diabetes, pDCs are epigenetically altered to increase IL-6 expression and direct the adaptive immune response toward pathologic TH17 CD4+ T-cell-mediated chronic inflammation and poor wound healing.

Amplified STAT3 signaling alters expression of CD39 in STAT3 gain-of-function (STAT3 GOF) and in healthy donor CD8 +T cells

Abstract It is still unknown whether primary immune regulatory disorders (PIRD) due to genetic variants that amplify T cell receptor and/or inflammatory cytokine signaling yield T cell exhaustion. Here, we investigate the impact of dysregulated cytokine signaling on CD8 +T cells in STAT3 gain-of-function (STAT3 GOF) patients. Immune profiling via 43-parameter CyTOF reveals altered CD8 +T cell differentiation, including loss of naïve cells, increased frequency of effector memory and T EMRAcells, and downregulation of markers of stemness (i.e., TCF-1 and CD127) in STAT3 GOF. Additionally, STAT3 GOF CD8 +T cells demonstrate decreased IL-2 production and proliferation. We identify increased expression of the ecto-enzyme, CD39, which has been reported on exhausted cells and hydrolyzes extracellular ATP, on CD8 +T cells from STAT3 GOF patients and in a mouse model of STAT3 GOF. To identify the signals regulating CD39, healthy donor CD8 +T cells were cultured with STAT3-activating cytokines alone but did not upregulate CD39. However, TCR engagement led to robust CD39 expression which was further augmented with STAT3-activating cytokines. Specifically, CD39 upregulation occurs in pSTAT3 Y705+cells. Finally, sorted CD39 +CD8 +T cells hydrolyzed ATP and suppressed effector CD8 +T cell proliferation compared to CD39 −CD8 +T cells. These data suggest that STAT3 signaling may play a role in regulating CD39 levels and thus contribute to CD8 +T cell exhaustion, which will be explored in future work. Understanding how dysregulated STAT3 signaling impacts T cell function in these rare patients can improve our understanding of this complex disease and may yield insights into shared aspects of T cell dysfunction found in more common inflammatory diseases. HHMI Gilliam Fellowship for Advanced Studies (GT15736) Penn Presidential Fellowship PIDTC (Henrickson Lab) IDF (Henrickson Lab) NIH NIAD K08AI135091 (Henrickson Lab) Burroughs Wellcome Fund CAMS (Henrickson Lab)

Paradoxical activation of chronic lymphocytic leukemia cells by ruxolitinib in vitro and in vivo.

Chronic lymphocytic leukemia (CLL) is characterized by an aberrant cytokine network that can support tumor growth by triggering janus kinase (JAK)/STAT pathways. Targeting cytokine-signaling should then be a rational therapeutic strategy but the JAK inhibitor ruxolitinib failed to control and seemingly accelerated the disease in clinical trials. The effect of ruxolitinib on primary human CLL cells was studied in vitro and in vivo. Ruxolitinib increased phosphorylation of IRAK4, an important toll-like receptor (TLR)- signaling intermediate, in circulating CLL cells in vitro. It also enhanced p38 and NFKB1 phosphorylation while lowering STAT3 phosphorylation in CLL cells activated with TLR-7/8 agonists and IL-2. Among the cytokines made by activated CLL cells, high levels of IL-10 contributed strongly to STAT3 phosphorylation and inhibited TLR7 activity. Ruxolitinib limited TLR-mediated IL10 transcription and markedly reduced IL-10 production in vitro. It also decreased blood levels of IL-10 while increasing TNFα along with phospho-p38 expression and gene sets associated with TLR-activation in CLL cells in vivo. The bruton's tyrosine kinase inhibitor ibrutinib decreased IL-10 production in vitro but, in contrast to ruxolitinib, blocked initial IL10 transcription induced by TLR-signaling in vitro, decreased TNFα production, and deactivates CLL cells in vivo. These findings suggest the possible benefits of inhibiting growth factors with JAK inhibitors in CLL are outweighed by negative effects on potential tumor suppressors such as IL-10 that allow unrestrained activation of NFκB by drivers such as TLRs. Specific inhibition of growth-promoting cytokines with blocking antibodies or infusing suppressive cytokines like IL-10 might be better strategies to manipulate cytokines in CLL.

FIBRONECTIN TYPE III DOMAIN CONTAINING 5 MITIGATES CHONDROCYTE SENESCENCE, SUBCHONDRAL OSTEOCLAST FORMATION, AND OSTEOARTHRITIS DEVELOPMENT THROUGH INHIBITING INTERLEUKIN-6

Senescent chondrocyte and subchondral osteoclast overburden aggravate inflammatory cytokine and pro-catabolic proteinase overproduction, accelerating extracellular matrix degradation and pain during osteoarthritis (OA). Fibronectin type III domain containing 5 (FNDC5) is found to promote tissue homeostasis and alleviate inflammation. This study aimed to characterize what role Fndc5 may play in chondrocyte aging and OA development.Serum and macroscopically healthy and osteoarthritic cartilage were biopsied from patients with knee OA who received total knee replacement. Murine chondrocytes were transfected with Fndc5 RNAi or cDNA. Mice overexpressing Fndc5 (Fndc5Tg) were operated to have destabilized medial meniscus mediated (DMM) joint injury as an experimental OA model. Cellular senescence was characterized using RT-PCR analysis of p16INK4A, p21CIP1, and p53 expression together with ß-galactosidase activity staining. Articular cartilage damage and synovitis were graded using OARSI scores. Osteophyte formation and mechanical allodynia were quantified using microCT imaging and von Frey filament, respectively. Osteoclast formation was examined using tartrate-resistant acid phosphatase staining.Senescent chondrocyte and subchondral osteoclast overburden together with decreased serum FNDC5 levels were present in human osteoarthritic cartilage. Fndc5 knockdown upregulated senescence program together with increased IL-6, MMP9 and Adamts5 expression, whereas Alcian blue-stained glycosaminoglycan production were inhibited. Forced Fndc5 expression repressed senescence, apoptosis and IL-6 expression, reversing proliferation and extracellular matrix production in inflamed chondrocytes. Fndc5Tg mice showed few OA signs, including articular cartilage erosion, synovitis, osteophyte formation, subchondral plate sclerosis and mechanical allodynia together with decreased IL-6 production and few senescent chondrocytes and subchondral osteoclast formation during DMM-induced joint injury. Mechanistically, Fndc5 reversed histone H3K27me3-mediated IL-6 transcription repression to reduce reactive oxygen species production.Fndc5 loss correlated with OA development. It was indispensable in chondrocyte growth and anabolism. This study sheds light onto the anti-ageing and anti-inflammatory actions of Fndc5 to chondrocytes; and highlights the chondroprotective function of Fndc5 to compromise OA.

Hyper‐IgE syndrome presenting with early life craniosynostosis in monozygotic twin sisters

<scp>Hyper‐IgE</scp> syndrome presenting with early life craniosynostosis in monozygotic twin sisters

Celastrol pretreatment attenuates concanavalin A-induced hepatitis in mice by suppressing interleukin-6/STAT3-interleukin-17 signaling.

Celastrol is extracted from Tripterygium wilfordii Hook F. It has been reported to have protective effects against various liver diseases and immune regulation of autoimmune diseases. However, little is known about whether celastrol protects against immune-mediated hepatitis. This study aimed to investigate the effect of celastrol on liver injury induced by concanavalin A (ConA) and the potential mechanisms. Intravenous administration of ConA was applied to induce acute liver injury in mice with or without pretreatment of celastrol. The effects of celastrol on ConA-induced liver injury were further demonstrated by biochemical and histopathological assessments, immunoblotting, and flow cytometry analysis. Both biochemical and histopathological observations showed that pretreatment of celastrol significantly ameliorated liver injury induced by ConA. Moreover, the hepatocyte apoptosis and inflammatory responses induced by ConA were also improved in celastrol-pretreated mice. Further studies revealed that these improvements were characterized as the celastrol-mediated suppression of total interleukin (IL)-17 from liver mononuclear cells in ConA-treated mice. Flow cytometry analysis suggested that celastrol specifically decreased IL-17 production by CD4+ T cells but not by CD8+ T cells. Fundamentally, pretreatment with celastrol inhibited both the IL-6 produced by F4/80+ macrophages and the IL-6 receptor on Th17 cells in the liver, which further led to the downregulated activation of STAT3, thus accounting for blocked Th17 signaling. Celastrol may exhibit immune regulatory effects by regulating IL-6/STAT3-IL-17 signaling in ConA-induced hepatitis, which suggested new potentials for celastrol to be applied in treating immune-mediated liver diseases.

Oestrogen and Vibration Improve Intervertebral Disc Cell Viability and Decrease Catabolism in Bovine Organ Cultures.

Postmenopausal women are at an increased risk for intervertebral disc degeneration, possibly due to the decrease in oestrogen levels. Low-magnitude, high-frequency vibration (LMHFV) is applied as a therapeutic approach for postmenopausal osteoporosis; however, less is known regarding possible effects on the intervertebral disc (IVD) and whether these may be oestrogen-dependent. The present study investigated the effect of 17β-oestradiol (E2) and LMHFV in an IVD organ culture model. Bovine IVDs (n = 6 IVDs/group) were treated with either (i) E2, (ii) LMHFV or (iii) the combination of E2 + LMHFV for 2 or 14 days. Minor changes in gene expression, cellularity and matrix metabolism were observed after E2 treatment, except for a significant increase in matrix metalloproteinase (MMP)-3 and interleukin (IL)-6 production. Interestingly, LMHFV alone induced cell loss and increased IL-6 production compared to the control. The combination of E2 + LMHFV induced a protective effect against cell loss and decreased IL-6 production compared to the LMHFV group. This indicates possible benefits of oestrogen therapy for the IVDs of postmenopausal women undergoing LMHFV exercises.