Frequency Of Th17 Cells Research Articles

Elevated profiles of peripheral Th22, Th17, Th2 cells, and decreased percentage of Th1 cells in breast cancer patients.

Th22 subset is a particular type of CD4+ T helper cells subset. Our study aimed to explore the expression level of circulating Th22, Th17, Th1, and Th2 cells and the possible mechanism of these cells in breast cancer (BC) with different pathological features. Our study enrolled 43 newly diagnosed BC patients and 30 healthy controls. Frequencies of peripheral Th22, Th17, Th1, and Th2 cells were tested by flow cytometry. Concentrations of IL-22 cytokine in plasma were examined by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was done to test aromatic hydrocarbon receptor (AHR) and RAR-associated orphan receptor C (RORC) gene expression. Frequencies of Th22, Th17, Th2 subsets, and the plasma IL-22 level was obviously higher in the BC patients. A positive correlation between Th22 frequency and IL-22 concentration in plasma was detected in BC patients. Furthermore, the percentage of Th22, Th2 subsets in peripheral blood of HER2 positive BC was higher than that in HER2 negative BC patients. A negative correlation between Th1 subset and Ki-67% as well as a positive correlation between Th2 subset and Ki-67% was found in BC patients. The proportion of Th1 cells in BC patients was significantly lower than that of the control group. Expression of AHR and RORC transcription factors were also observed to be upregulated in the BC patients. Furthermore, Th22 cells were positively correlated with BC tumor stage and clinical outcomes. The BC patients with a higher percentage of Th22, Th17, Th1 cells or a lower percentage of Th1 cells showed a decreased trend of survival rate. Th22, Th17, Th1, and Th2 subsets may play an essential role in BC patients. Th22, Th17, Th1, and Th2 cells may have potential significance to be used as clinical markers in BC patients with different molecular classification. Th22 cells may have potential value in BC patients' outcomes prediction, providing clinical value.

T helper cell responses in adult diarrheal patients following natural infection with enterotoxigenic Escherichia coli are primarily of the Th17 type.

Infection with enterotoxigenic Escherichia coli (ETEC) gives rise to IgA antibodies against both the heat labile toxin (LT) and colonization factors (CFs), which are considered to synergistically protect against ETEC diarrhea. Since the development of ETEC-specific long lived plasma cells and memory B cells is likely to be dependent on T helper (Th) cells, we investigated if natural ETEC diarrhea elicits ETEC-specific Th cells and their relation to IgA responses. Th cell subsets were analyzed in adult Bangladeshi patients hospitalized due to ETEC diarrhea by flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) isolated from blood collected day 2, 7, 30 and 90 after hospitalization as well as in healthy controls. The LT- and CF-specific Th responses were determined by analysis of IL-17A and IFN-γ in antigen stimulated PBMC cultures using ELISA. ETEC-specific IgA secreted by circulating antibody secreting cells (plasmablasts) were analyzed by using the antibodies in lymphocyte supernatants (ALS) ELISA-based method and plasma IgA was also measured by ELISA. ETEC patients mounted significant ALS and plasma IgA responses against LTB and CFs on day 7 after hospitalization. ETEC patients had significantly elevated proportions of memory Th cells with a Th17 phenotype (CCR6+CXCR3-) in blood compared to controls, while frequencies of Th1 (CCR6-CXCR3+) or Th2 (CCR6-CXCR3-) cells were not increased. Antigen stimulation of PBMCs revealed IL-17A responses to LT, most clearly observed after stimulation with double mutant heat labile toxin (dmLT), but also with LT B subunit (LTB), and to CS6 in samples from patients with LT+ or CS6+ ETEC bacteria. Some individuals also mounted IFN-γ responses to dmLT and LTB. Levels of LTB specific IgA antibodies in ALS, but not plasma samples correlated with both IL-17A (r=0.5, p=0.02) and IFN-γ (r=0.6, p=0.01) responses to dmLT. Our results show that ETEC diarrhea induces T cell responses, which are predominantly of the Th17 type. The correlations between IL-17A and IFN-g and intestine-derived plasmablast responses support that Th responses may contribute to the development of protective IgA responses against ETEC infection. These observations provide important insights into T cell responses that need to be considered in the evaluation of advanced ETEC vaccine candidates.

A newly developed PLD1 inhibitor ameliorates rheumatoid arthritis by regulating pathogenic T and B cells and inhibiting osteoclast differentiation

Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays multiple roles in inflammation. We investigated the therapeutic effects of the newly developed PLD1 inhibitors A2998, A3000, and A3773 in vitro and in vivo rheumatoid arthritis (RA) model. A3373 reduced the levels of LPS-induced TNF-α, IL-6, and IgG in murine splenocytes in vitro. A3373 also decreased the levels of IFN-γ and IL-17 and the frequencies of Th1, Th17 cells and germinal-center B cells, in splenocytes in vitro. A3373 ameliorated the severity of collagen-induced arthritis (CIA) and suppressed infiltration of inflammatory cells into the joint tissues of mice with CIA compared with vehicle-treated mice. Moreover, A3373 prevented systemic bone demineralization in mice with CIA and suppressed osteoclast differentiation and the mRNA levels of osteoclastogenesis markers in vitro. These results suggest that A3373 has therapeutic potential for RA.

Fasudil and SR1001 synergistically protect against sepsis-associated pancreatic injury by inhibiting RhoA/ROCK pathway and Th17/IL-17 response

Sepsis is defined as a dysregulated host response to infection that can result in organ dysfunction and high mortality, which needs more effective treatment urgently. Pancreas is one of the most vulnerable organs in sepsis, resulting in sepsis-associated pancreatic injury, which is a fatal complication of sepsis. The aim of this study was to investigate the effect of combination of fasudil and SR1001 on sepsis-associated pancreatic injury and to explore the underlying mechanisms. The model of sepsis-associated pancreatic injury was induced by cecal ligation and puncture. Pancreatic injury was evaluated by HE staining, histopathological scores and amylase activity. The frequency of Th17 cells was analyzed by flow cytometry. Serum IL-17 level was determined by ELISA. Protein levels of RORγt, p-STAT3, GEF-H1, RhoA and ROCK1 were determined by Western blot. The apoptosis of pancreatic cells was examined by TUNEL analysis and Hoechst33342/PI staining. Compared to the sham group, the model group showed significant pathological injury including edema, hyperemia, vacuolization and necrosis. After treatment with fasudil, model mice showed an obvious reduction of Th17 cells and IL-17. SR1001 significantly reduced the expressions of GEF-H1, RhoA and ROCK1 in the model mice. The combination treatment with fasudil and SR1001 significantly inhibited the differentiation of Th17 cells, expressions of IL-17, GEF-H1, RhoA and ROCK1, which were more effective than each mono-treatment. In addition, our data revealed a remarkable decrease of apoptosis in pancreatic acinar cells culturing with fasudil or SR1001, which was further inhibited by their combination culture. Lipopolysaccharide remarkably upregulated the differentiation of Th17 cells in vitro, which could be significantly downregulated by fasudil or SR1001, and further downregulated by their combination treatment. Taken together, the combination of fasudil with SR1001 has a synergistic effect on protecting against sepsis-associated pancreatic injury in C57BL/6 mice.

Fc multimers effectively treat murine models of multiple sclerosis.

Multiple Sclerosis (MS) is a chronic neurodegenerative disease with limited therapeutic options. Recombinant Fc multimers (rFc), designed to mirror many of the anti-inflammatory activities of Intravenous Immunoglobulin (IVIG), have been shown to effectively treat numerous immune-mediated diseases in rodents. In this study we used the experimental autoimmune encephalomyelitis (EAE) murine model of MS to test the efficacy of a rFc, M019, that consists of multimers of the Fc portion of IgG2, in inhibiting disease severity. We show that M019 effectively reduced clinical symptoms when given either pre- or post-symptom onset compared to vehicle treated EAE induced mice. M019 was effective in reducing symptoms in both SJL model of relapsing remitting MS as well as the B6 model of chronic disease. M019 binds to FcγR bearing-monocytes both in vivo and in vitro and prevented immune cell infiltration into the CNS of treated mice. The lack of T cell infiltration into the spinal cord was not due to a decrease in T cell priming; there was an equivalent frequency of Th17 cells in the spleens of M019 and vehicle treated EAE induced mice. Surprisingly, there was an increase in chemokines in the sera but not in the CNS of M019 treated mice compared to vehicle treated animals. We postulate that M019 interacts with a FcγR rich monocyte intermediary to prevent T cell migration into the CNS and demyelination.

Elevated circulating CD19+CD24hiCD38hi B cells display pro-inflammatory phenotype in idiopathic membranous nephropathy

CD19+CD24hiCD38hi regulatory B cells exert immunosuppressive functions by producing IL-10, but their role in idiopathic membranous nephropathy (IMN) remains elusive. Here, we investigated the frequency and functional changes of circulating CD19+CD24hiCD38hi B cells and evaluated the correlation of CD19+CD24hiCD38hi B cells with clinical features and T helper cell subsets in IMN patients. Compared with healthy controls (HCs), IMN patients showed an increased frequency of CD19+CD24hiCD38hi B cells, but a significant reduction in the percentage of CD19+CD24hiCD38hi B cells was observed 4 weeks after cyclophosphamide treatment. The frequency of CD19+CD24hiCD38hi B cells was positively correlated with the levels of 24h urinary protein, but negatively correlated with serum total protein and serum albumin, respectively. CD19+CD24hiCD38hi B cells in IMN patients displayed a skewed pro-inflammatory cytokine profile with a higher level of IL-6 and IL-12, but a lower concentration of IL-10 than their healthy counterparts. Accompanied by upregulation of Th2 and Th17 cells in IMN patients, the percentage of CD19+CD24hiCD38hi B cell subset was positively associated with Th17 cell frequency. In conclusion, CD19+CD24hiCD38hi B cells were expanded but functionally impaired in IMN patients. Their altered pro-inflammatory cytokine profile may contribute to the pathogenesis of IMN.

Increased circulating Th17 cell populations in patients with pancreatic ductal adenocarcinoma.

T-helper 17 (Th17) cells are a subset of CD4+ helper T cells that produce interleukin 17 (IL-17) and play a crucial role in the pathogenesis of inflammatory and autoimmune diseases. Few studies have been conducted to determine the role of Th17 cells in the tumorigenesis and development of pancreatic ductal adenocarcinoma (PDAC); however, its role is still unclear. In this study, the percentage of circulating Th17 cells and serum levels of IL-17A and IL-23 were analyzed using flow cytometry and ELISA, respectively, in 40 PDAC patients, 30 chronic pancreatitis (CP) patients and 30 healthy controls (HC). In addition, the mRNA expression levels of IL-17A, STAT3 and RORγt in tissue samples were quantified by qRT-PCR. The results showed that the percentage of circulating Th17 cells and the concentrations of serum IL-17A and IL-23 were significantly increased in PDAC patients as compared to CP and HC (P < 0.001). In addition, the higher level of IL-17A was significantly correlated with the poor overall survival of the PDAC patients. Furthermore, the frequencies of Th17 cells and IL-17A were significantly higher in stage III+IV PDAC patients versus stage I+II. A significant increase in IL-17A, STAT3 and RORγT mRNA was observed in patients with PDAC. Taken together, these findings suggest that the increased circulating Th17 cells and serum IL-17A may be involved in the development and metastasis of PDAC, and thus represent potential targets for the treatment of PDAC.

Percentage of Th1 and Th17 cells and serum level of IL-17 and IFN-γ cytokines in COVID-19-associated mucormycosis.

The considerable number of the 2019 coronavirus disease (COVID-19) patients who developed mucormycosis infections in West and Central Asia urged a need to investigate the underlying causes of this fatal complication. It was hypothesized that an immunocompromised state secondary to the excessive administration of anti-inflammatory drugs was responsible for the outburst of mucormycosis in COVID-19 patients. Therefore, we aimed to study the implication of two major subsets of adaptive immunity T helper (Th)-1 and Th17 cells in disease development. Thirty patients with COVID-19-associated mucormycosis, 38 with COVID-19 without any sign or symptom of mucormycosis, and 26 healthy individuals were included. The percentage of Th1 and Th17 cells in peripheral blood, as well as the serum levels of interleukin (IL)-17 and interferon-gamma (IFN-γ), were evaluated using flow cytometry and ELISA techniques, respectively. Th17 cell percentage in patients with COVID-19-associated mucormycosis was significantly lower than in COVID-19 patients (P-value: <0.001) and healthy subjects (P-value: 0.01). In addition, the serum level of IL-17 in COVID-19 patients was significantly higher than that of healthy individuals (P-value: 0.01). However, neither the frequency of Th1 cells nor the serum level of IFN-γ was different between the study groups. Given the critical role of Th17 cells in the defense against mucosal fungal infections, these findings suggest that low numbers of Th17 and insufficient levels of IL-17 might be a predisposing factor for the development of mucormycosis during or after COVID-19 infection.

Retinoic Acid-Related Orphan Receptor α Is Required for Generation of Th2 Cells in Type 2 Pulmonary Inflammation.

The transcription factor retinoic acid-related orphan receptor α (RORα) is important in regulating several physiological functions, such as cellular development, circadian rhythm, metabolism, and immunity. In two invivo animal models of type 2 lung inflammation, Nippostrongylus brasiliensis infection and house dust mite (HDM) sensitization, we show a role for Rora in Th2 cellular development during pulmonary inflammation. N. brasiliensis infection and HDM challenge induced an increase in frequency of Rora-expressing GATA3+CD4 T cells in the lung. Using staggerer mice, which have a ubiquitous deletion of functional RORα, we generated bone marrow chimera mice, and we observed a delayed worm expulsion and reduced frequency in the expansion of Th2 cells and innate lymphoid type 2 cells (ILC2s) in the lungs after N. brasiliensis infection. ILC2-deficient mouse (Rorafl/flIl7raCre) also had delayed worm expulsion with associated reduced frequency of Th2 cells and ILC2s in the lungs after N. brasiliensis infection. To further define the role for Rora-expressing Th2 cells, we used a CD4-specific Rora-deficient mouse (Rorafl/flCD4Cre), with significantly reduced frequency of lung Th2 cells, but not ILC2, after N. brasiliensis infection and HDM challenge. Interestingly, despite the reduction in pulmonary Th2 cells in Rorafl/flCD4Cre mice, this did not impact the expulsion of N. brasiliensis after primary and secondary infection, or the generation of lung inflammation after HDM challenge. This study demonstrates a role for RORα in Th2 cellular development during pulmonary inflammation that could be relevant to the range of inflammatory diseases in which RORα is implicated.

Temporal dynamics of Th17 responses to Candida albicans in rheumatoid arthritis: Implications for immunity and treatment

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by symmetrical polyarthritis and systemic inflammation. Recent studies have highlighted the critical role of the Th17/IL-17 axis in RA pathogenesis, with elevated Th17 cell frequencies and IL-17 production contributing to disease progression. Candida albicans, a commensal fungus residing in mucocutaneous surfaces, relies on Th17-mediated immunity for protection against mucocutaneous candidiasis. However, RA patients exhibit impaired Th17 responses, increasing their susceptibility to C. albicans infections. This review examines the temporal dynamics of Th17 responses to C. albicans in RA patients, analyzing the expression trends of Th17 and regulatory T cells (Tregs) over 12, 24, and 48 hours. Findings indicate that RA patients have a diminished initial Th17 response, inadequate IL-17 production at peak activity, and sustained immune dysfunction, leading to compromised fungal clearance. Additionally, biologic therapies targeting the Th17/IL-17 pathway, while effective for RA symptom management, may exacerbate infection risks. Understanding these dynamics is crucial for developing strategies that enhance antifungal immunity without aggravating RA symptoms. Further research is essential to optimize treatment protocols that balance antifungal defenses and autoimmune regulation.

Vitamins A and D Enhance the Expression of Ror-γ-Targeting miRNAs in a Mouse Model of Multiple Sclerosis.

Autoreactive T cells, particularly those characterized by a Th17 phenotype, exert significant influence on the pathogenesis of multiple sclerosis (MS). The present study aimed to elucidate the impact of individual and combined administration of vitamin A and D on neuroinflammation, and microRNAs (miRNAs) involved in T helper (Th)17 development, utilizing a murine model of experimental autoimmune encephalomyelitis (EAE). EAE was induced in C57BL/6 mice, and 3 days prior to immunization, intraperitoneal injections of vitamins A and D or their combination were administered. Th17 cell percentages were determined in splenocytes utilizing intracellular staining and flow cytometry. Furthermore, the expression of Ror γ-t, miR-98-5p and Let-7a-5p, was measured in both splenocytes and spinal cord tissues using RT-PCR. Treatment with vitamin A and D resulted in a reduction in both disease severity in EAE mice. Treated mice showed a decreased frequency of Th17 cells and lower expression levels of IL17 and Ror γ-t in splenocytes and spinal cord. The spinal cord tissues and splenocytes of mice treated with vitamins A, D, and combined A+D showed a significant upregulation of miR-98-5p and Let-7a-5p compared to the EAE group. Statistical analysis indicated a strong negative correlation between miR-98-5p and Let-7a-5p levels in splenocytes and Ror-t expression. Our findings indicate that the administration of vitamins A and D exerts a suppressive effect on neuroinflammation in EAE that is associated with a reduction in the differentiation of T cells into the Th17 phenotype and is mediated by the upregulation of miR-98-5p and Let-7a-5p, which target the Ror γ-t.

#4983 THE UNEXPECTABLE BALANCE OF PROINFLAMMATORY CELLS IN PERITONEAL DIALYSIS PATIENTS

Abstract Background and Aims Chronic kidney disease (CKD) is a complex and heterogeneous disease and is increasingly becoming a global public health concern. It is is characterized by a persistent inflammation state which appear to be caused by cytokines, grown factors and hormones. Peritoneal dialysis (PD) accounts for 9% of all kidney replacement therapy (KRT) and over time, some changes are expected to occur in the peritoneal membrane mainly due to exposure to PD solutions, infectious processes and non-infectious complications. This inflammatory process is poorly understood and needs further clarification, but Th17 cells and Th1 cells seem to be involved. Therefore, we aimed to quantify the frequency of peripheral immune cells and the concentration of IL-17A in peripheral blood (PB) and peritoneal effluent (PE) and compare to a healthy control group. Method In a cross-sectional study of 26 PD patients and 10 healthy, age and sex-matched controls we evaluated PB and PE frequencies of T lymphocytes, T CD4, T CD8 and Th1 cells by flow cytometry as well as IL-17 concentrations using enzyme-linked immunosorbent assay. Comparisons were performed between PD patients and controls and between the different groups of PD patients according to PD modality, number of exchanges, dialysate tonicity, extraneal use, infusion and daily total volumes, type of peritoneal transport and volume indexes. Statistical analyses were performed using the SPSS statistics version 26. Results Table 1 summarizes the main characteristics of PD patients and controls. Overall, in comparison with the control group, the frequency of Th1 cells in PB of PD patients was significantly lower (P = 0.001). On the other hand, frequency of Th1 cells substantially expanded in the PE, as compared to the PB (P = 0.001). PD patients also presented lower Th17 cells frequencies (P = 0.061) and those prescribed Extraneal had the lowest frequencies of Th17 (P = 0.030). IL-17A concentration was significantly higher in PE vs PB of PD patients (P = 0.012). Finally, the frequency of total lymphocytes in high PD transporters was significantly higher in comparison with high-average transporters (P = 0.021). Conclusion In our cohort, PE presented high concentrations of proinflammatory cells and cytokines. Th1 cells rather than Th17 cells were increased, pointing to a predominant cytolytic activity but IL-17 was also increased. Treatment with Extraneal associated with a lower frequency of these cells, somehow counterbalancing the inflammatory millieu associated with high glucose concentrations.

Targeting the NLRP3 inflammasome reduces inflammation in hidradenitis suppurativa skin.

Treatment for the debilitating disease hidradenitis suppurativa (HS) is inadequate in many patients. Despite an incidence of approximately 1%, HS is often underrecognized and underdiagnosed and is associated with a high morbidity and poor quality of life. A better understanding of its pathogenesis is required to design new therapeutic strategies. In this study we employed single cell RNA sequencing to analyse gene expression in immune cells isolated from involved HS skin compared with healthy skin. Flow cytometry was used to quantify the absolute numbers of the main immune populations. Secretion of inflammatory mediators from skin explant cultures was measured using multiplex assays and ELISA. Single cell RNA sequencing analysis identified a significant enrichment in the frequency of plasma cells, Th17 cells and dendritic cell subsets in HS skin, and the immune transcriptome was distinct and much more heterogenous when compared with healthy skin. Flow cytometry revealed significantly increased numbers of T cells, B cells, neutrophils, dermal macrophages, and dendritic cells into involved HS skin. Genes and pathways associated with Th17 cells, IL-17, IL-1β, and the NLRP3 inflammasome were enhanced in HS skin, particularly in samples with a high inflammatory load. Inflammasome constituent genes principally mapped to Langerhans cells and a subpopulation of dendritic cells. The secretome of HS skin explants contained significantly increased concentrations of inflammatory mediators including IL-1β and IL-17A, and culture with an NLRP3 inflammasome inhibitor significantly reduced the secretion of these as well as other key mediators of inflammation. These data provide a rationale for targeting the NLRP3 inflammasome in HS using small molecule inhibitors which are currently being tested for other indications.

Staphylococcus aureus biofilm properties and chronic rhinosinusitis severity scores correlate positively with total CD4+ T-cell frequencies and inversely with its Th1, Th17 and regulatory cell frequencies.

Chronic rhinosinusitis (CRS) represents chronic inflammation of the sinus mucosa characterised by dysfunction of the sinuses' natural defence mechanisms and induction of different inflammatory pathways ranging from a Th1 to a Th2 predominant polarisation. Recalcitrant CRS is associated with Staphylococcus aureus dominant mucosal biofilms; however, S. aureus colonisation of the sinonasal mucosa has also been observed in healthy individuals challenging the significance of S. aureus in CRS pathogenesis. We aimed to investigate the relationship between CRS key inflammatory markers, S. aureus biofilm properties/virulence genes and the severity of the disease. Tissue samples were collected during endoscopic sinus surgery from the ethmoid sinuses of CRS patients with (CRSwNP) and without (CRSsNP) nasal polyps and controls (n = 59). CD3+ T-cell subset frequencies and key inflammatory markers of CD4+ helper T cells were determined using FACS analysis. Sinonasal S. aureus clinical isolates were isolated (n = 26), sequenced and grown into biofilm in vitro, followed by determining their properties, including metabolic activity, biomass, colony-forming units and exoprotein production. Disease severity was assessed using Lund-Mackay radiologic scores, Lund-Kennedy endoscopic scores and SNOT22 quality of life scores. Our results showed that S. aureus biofilm properties and CRS severity scores correlated positively with total CD4+ T-cell frequencies but looking into CD4+ T-cell subsets showed an inverse correlation with Th1 and Th17 cell frequencies. CD4+ T-cell frequencies were higher in patients harbouring lukF.PV-positive S. aureus while its regulatory and Th17 cell subset frequencies were lower in patients carrying sea- and sarT/U-positive S. aureus. Recalcitrant CRS is characterised by increased S. aureus biofilm properties in relation to increased total CD4+ helper T-cell frequencies and reduced frequencies of its Th1, Th17 and regulatory T-cell subsets. These findings offer insights into the pathophysiology of CRS and could lead to the development of more targeted therapies.

Examining methotrexate’s safety and efficacy in combination with immune checkpoint inhibitors to control immune related inflammatory arthritis in cancer

Abstract Immune checkpoint inhibitors (ICIs) are associated with high rates of toxic side effects known as immune related adverse events such as inflammatory arthritis (ir-RA). ir-RA is empirically treated with drugs for rheumatoid arthritis such as methotrexate (MTX) and hydroxychloroquine which was recently shown to reduce the anti-tumor benefit of ICIs in mouse models of cancer. We have set out to determine the safety of MTX use in combination with ICIs, its effect on the anti-tumoral immune response and its efficacy in treating ir-RA in a novel arthritis-B16 PD-L1 tumor mouse model. We observed that MTX did not diminish the anti-tumor benefit of ICIs in our melanoma model and significantly altered the intra-tumoral CD8 +T cell population by promoting the formation of central memory cells and diminishing PD1 expression. We have identified that MTX acts on neuropilin-1 (NRP1), a receptor identified as a CD8 +T cell exhaustion marker promoting terminal exhaustion and limiting memory cell formation in tumors. We have shown that MTX treatment in vitro reduces the expression of NRP1 on stimulated CD8 +T cells. We have also advanced in the development of a novel arthritis-tumor model showing that MTX can control and diminish inflammation in the delayed type hypersensitivity arthritis (DTHA) and reduced the frequency of Th1 and Th17 cells in the popliteal lymph nodes of mice, with Th17 cells known to have a crucial role in ir-RA. Together these results show that MTX is safe for use in combination with ICIs in our tumor model and is effective in our DTHA model that mimics the immune profile of ir-RA. With the development of our combined arthritis-tumor model we will be able to assess MTX’s efficacy in controlling arthritis in ICIs treated tumor bearing mice.

DJ-X-013 ameliorates inflammation by reducing TNF-α/NF-κB expression and experimental colitis by inducing myeloid-derived suppressor cells

Abstract Inflammation is a body’s defense response for a short duration however if persists for long periods, it causes tissue damage and pathological consequences. Moreover, it paves the way for several diseases including inflammatory bowel disease (IBD). Therefore, there is an urgent need for a natural and safe anti-inflammatory therapeutic compound. To address this issue, we aimed to determine the anti-inflammatory effects of DJ-X-013, using lipopolysaccharide (LPS) activated RAW 264.7 macrophage in vitro and dextran sodium sulfate (DSS) induced experimental model of colitis in vivo. DJ-X-013 drastically reduced tumor necrosis factor-alpha and inducible nitric oxide synthase expression in LPS-activated RAW 264.7 macrophages. Further, DJ-X-013 also impeded the expression of NF-κB and other inflammatory markers and pathways like IL-6, IL-1β, STAT3, ERK, and JNK as compared to LPS-activated cells. Additionally, DJ-X-013 also modulates the migratory activity of RAW 264.7 macrophages during wound healing. Next, using DSS induced colitis model, we observed that the reduction of body weight and colon length were slightly improved in the DJ-X-013 group compared to the control. Intriguingly, myeloid-derived suppressor cells, neutrophils, and CD8 +T cells increased, while Th17 cells frequency decreased in the spleen, mesenteric lymph nodes, and colon lamina propria in DJ-X-013 treated group as compared to DSS. However, natural killer and natural killer T cells were marginally enhanced in the DJ-X-013 group as compared to the DSS alone. Hence, the DJ-X-013 exhibits an immunomodulatory effect and might be a promising drug for IBD and other inflammatory diseases, however, further investigation is required for prudent conclusions. This research is supported by NIH grant R01 AI140405

Abstract 5116: Inflammasome unleashing during anti-PD-1 therapy modulates CD8+ T cell exhaustion through Th17 cells

Abstract PD-1/PD-L1 blockade has greatly improved overall and progression free survival in a variety of cancers. However, only a minority of patients with advanced disease benefit from this approach. We have recently showed that inflammasome unleashing by inhibition of the intracellular cation channel TMEM176B with Boritinib (BayK8644) augments anti-PD-1 efficacy. However, the mechanisms involved were not completely understood. We hypothesized that inflammasome activation triggered by Boritinib may recruit Th17 cells to modulate CD8+ T cell exhaustion. To test this hypothesis, we have analyzed Th17 cells and subsets of exhausted CD8+ T cells in the tumor microenvironment of WT mice treated with anti-PD1 ± Boritinib. Combining Boritinib with anti-PD-1 led to an increased frequency of Th17 cells in the tumor microenvironment compared to anti-PD-1 alone. Furthermore, CD73 expression was down-regulated whereas IFN-γ was up-regulated in Th17 cells in the tumor microenvironment of Boritinib + anti-PD-1 treated mice, suggesting an effector phenotype of these cells. In addition, combination of anti-PD-1 with Boritinib results in an increased frequency of progenitor exhausted CD8+ T cells (CD44+PD-1+TCF1+TOX+) and transitory exhausted CD8+ T cells (CD44+PD-1+TCF1−TOX−CX3CR1+) in the tumor microenvironment compared to anti-PD-1 alone. Similar results were obtained using Tmem176b−/− mice treated with anti-PD-1, suggesting an on-target effect of Boritinib. To assess a potential role of Th17 cells in the anti-tumor immunity triggered by Boritinib + anti-PD-1, we treated WT and Il17a−/− mice. We found that Il17 is necessary for the anti-tumor effect of Boritinib + anti-PD-1, since Il17a−/− mice showed a reduced overall survival compared to WT mice. Under this combined therapy, IL-17 may impact on exhausted CD8+ T cells subsets since we found that transitory and terminally exhausted CD8+ T cells in the tumor microenvironment of Il17a−/− mice had less granzyme B than in WT mice. To directly show the role of Th17 cells in the anti-PD-1 anti-tumor immunity we adoptively transferred in vitro differentiated effector Th17 cells. Adoptive transfer of effector Th17 cells into tumor-bearing mice treated with anti-PD-1 resulted in better survival than mice treated with anti-PD-1 alone. To characterize the downstream mechanisms, we performed an in vivo cytotoxicity assay. We found that mice adoptively transferred with effector Th17 cells exhibited greater cytotoxicity by CD8+ T cells than mice treated with anti-PD-1 alone. Furthermore, this could be explained by an increased frequency in progenitor exhausted CD8+ T cells. Thus, effector Th17 cells potentiate CD8+ T cells on anti-PD-1 treatment. In conclusion, combination of anti-PD-1 with Boritinib augments the efficacy of the former by unleashing the inflammasome which results in an increased frequency of Th17 cells leading to modulation of exhausted CD8+ T cells. Citation Format: Sofia Russo, Mateo Malcuori, David Charbonnier, Mercedes Segovia, Marcelo Hill. Inflammasome unleashing during anti-PD-1 therapy modulates CD8+ T cell exhaustion through Th17 cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5116.

Anti-inflammatory and immunomodulatory effects of polysaccharide extracted from Wuguchong (maggot) on 2,4-dinitrochlorobenzene-induced atopic dermatitis in mice.

Atopic dermatitis (AD) is an inflammatory, heterogeneous, chronic skin disorder characterized by recurrent eczematous lesions and intense pruritus, and the pathophysiology mechanism of AD is known for immune dysregulation and inflammatory responses. Wuguchong (maggot) has been widely used in the wound field and found with pharmacological properties of the anti-inflammatory and immunomodulatory function. Recently, some polysaccharides were proven to have beneficial effects on AD skin lesions in mice and humans. However, the effect of the polysaccharide extracted from Wuguchong (PEW) on AD remains to be investigated. In the present study, we examined the anti-inflammatory and immunomodulatory effects of PEW on AD and explored the potential mechanisms. Balb/c mice were orally administrated with PEW to evaluate the therapeutic effect of PEW on 2,4-dinitrochlorobenzene (DNCB)-induced AD. Oral PEW administration significantly ameliorated the lesions and symptoms in AD mice, such as the ear thickness and ear swelling degree, epidermal and dermal thickness, and the infiltration of mast cells. In addition, PEW treatment decreased the levels of serum IgE and histamine, the frequencies of Th1 and Th17 cells, as well as the mRNA expression levels of Th1 and Th17 cytokines and nuclear transcript factors (IFN-γ, T-bet, IL-17A, and ROR-rt). Furthermore, the activation of the NF-κB pathway and the phosphorylation of MAPKs (p38, ERK, and JNK) were significantly suppressed by PEW treatment. Taken together, our study suggests that PEW exerts anti-inflammatory and immunomodulatory effects through inhibition of Th1 and Th17 responses and downregulation of NF-κB and MAPK pathways, PEW would be developed as a promising immune therapy for AD.

Pretreatment Frequency of Circulating Th17 Cells and FeNO Levels Predicted the Real-World Response after 1 Year of Benralizumab Treatment in Patients with Severe Asthma.

Benralizumab treatment reduces exacerbations and improves symptom control and quality of life in patients with severe eosinophilic asthma. However, the determination of biomarkers that predict therapeutic effectiveness is required for precision medicine. Herein, we elucidated the dynamics of various parameters before and after treatment as well as patient characteristics predictive of clinical effectiveness after 1 year of benralizumab treatment in severe asthma in a real-world setting. Thirty-six patients with severe asthma were treated with benralizumab for 1 year. Lymphocyte subsets in peripheral blood samples were analyzed using flow cytometry. Treatment effectiveness was determined based on the ACT score, forced expiratory volume in 1 s (FEV1), and the number of exacerbations. Benralizumab provided symptomatic improvement in severe asthma. Benralizumab significantly decreased peripheral blood eosinophil and basophil counts and the frequencies of regulatory T cells (Tregs), and increased the frequencies of Th2 cells. To our knowledge, this is the first study to show benralizumab treatment increasing circulating Th2 cells and decreasing circulating Tregs. Finally, the ROC curve to discriminate patients who achieved clinical effectiveness of benralizumab treatment revealed that the frequency of circulating Th17 cells and FeNO levels might be used as parameters for predicting the real-world response of benralizumab treatment in patients with severe asthma.

Anti-inflammatory Mechanisms of IL-38 in Chinese Patients with Allergic Rhinitis.

T-helper 17 (Th17) cell response is engaged in the onset of allergic rhinitis (AR). Moreover, interleukin (IL)-38 is thought to be involved in inhibiting cytokine secretion in the Th17 pathway. To evaluate the regulatory function of IL-38 on abnormal Th17 responses in Chinese patients with AR. Forty-five participants, divided into an AR group (n=25) and a control group (n=20), were recruited for the study. In addition, the expression of IL-38 and Th17-related cytokines was measured as well as the Th17 cell count in participants. By implementing recombinant IL-38 (rIL-38), the intervention of human peripheral blood mononuclear cells (PBMCs) was performed. Then, flow cytometry, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) were used to detect the Th17 milieu. The expression of IL-38 in the AR group notably reduced compared with that in the control, whereas Th17 cell frequency and the expression levels of its transcription factor (RORC) and cytokines (IL-17A and IL-23) increased. The differentiation and immune function of Th17 cells in PBMCs were inhibited by rIL-38. Th17 responses are inhibited by IL-38 in patients with AR. Therefore, the obtained findings indicate that IL-38 is a potential therapeutic target for Chinese patients with AR.