Induction Of TNF-α Research Articles

Identification of Functional Immune Biomarkers in Breast Cancer Patients.

Cancer immunotherapy has emerged as an effective, personalized treatment for certain patients, particularly for those with hematological malignancies. However, its efficacy in breast cancer has been marginal-perhaps due to cold, immune-excluded, or immune-desert tumors. Natural killer T (NKT) cells play a critical role in cancer immune surveillance and are reduced in cancer patients. Thus, we hypothesized that NKT cells could serve as a surrogate marker for immune function. In order to assess which breast cancer patients would likely benefit from immune cell-based therapies, we have developed a quantitative method to rapidly assess NKT function using stimulation with artificial antigen presenting cells followed by quantitative real-time PCR for IFN-γ. We observed a significant reduction in the percentage of circulating NKT cells in breast cancer patients, compared to healthy donors; however, the majority of patients had functional NKT cells. When we compared BC patients with highly functional NKT cells, as indicated by high IFN-γ induction, to those with little to no induction, following stimulation of NKT cells, there was no significant difference in NKT cell number between the groups, suggesting functional loss has more impact than physical loss of this subpopulation of T cells. In addition, we assessed the percentage of tumor-infiltrating lymphocytes and PD-L1 expression within the tumor microenvironment in the low and high responders. Further characterization of immune gene signatures in these groups identified a concomitant decrease in the induction of TNFα, LAG3, and LIGHT in the low responders. We next investigated the mechanisms by which breast cancers suppress NKT-mediated anti-tumor immune responses. We found that breast cancers secrete immunosuppressive lipids, and treatment with commonly prescribed medications that modulate lipid metabolism, can reduce tumor growth and restore NKT cell responses.

Liposome encapsulating pine bark extract in Nile tilapia: Targeting interrelated immune and antioxidant defense to combat coinfection with Aeromonas hydrophila and Enterococcus faecalis

Application of smart delivery systems for encapsulation of natural ingredients provides novel avenues and is being frequently developed. Thus, we aimed to highlight the effects of cyclosome liposomal pine bark extract (CL-PBE) on Nile tilapia growth, immunomodulation, antioxidant capacity and resistance against coinfection with Aeromonas hydrophila and Enterococcus faecalis and their associated virulence genes. The experiment was conducted on four fish groups receiving a control diet (control) along with three baseline meals supplemented with 200, 400 and 600 mg/kg diet of CL-PBE (CL-PBE 200, 400 and 600, respectively). At the end of the 12-weeks feeding trial, the tilapias were intraperitoneally challenged with virulent A. hydrophila strain and five days later, E. faecalis challenge was carried out. The results revealed that tilapias fed diets fortified with CL-PBE displayed significantly enhanced growth rate and feed conversion ratio in a dose-dependent manner. Moreover, we demonstrated that CL-PBE had potent antioxidant property presented by modulation of several markers of oxidative stress; substantial reductions in reactive oxygen species, hydrogen peroxide and malondialdehyde levels, an elevation in total antioxidant capacity and boosting glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) activities in fish serum and muscle tissues. This was also correlated with augmenting the expression of CAT, SOD, GSH-Px, Nrf2 and caspase-1 genes alongside reducing those of COX-2, HSP70 and iNOS genes in response to CL-PBE. Our data demonstrated that CL-PBE fortification counteracted the overly pronounced inflammatory response-mediated induction of IL-1β, TNF-α, MHCII and TLR2 genes at the transcriptional levels post coinfection together with promotion in MUC2 and IL-10 genes expression. Notably, our findings displayed optimal well-functioning fish immune system post dietary supplementation of CL-PBE for the protection against coinfection with A. hydrophila and E. faecalis. This was evident from the decline of their counts and hence encompassing the capacity to reduce cumulative mortality percentage in conjunction with interference with their virulence via the significant downregulatory effects of CL-PBE on E. faecalis esp and gelE and A. hydrophila act and fla virulence genes. Taken together, our study strongly suggested dietary inclusion of CL-PBE for Nile tilapias with superior growth performance and significant economic benefits coupled with potent stimulatory effects on antioxidant capacity and immune response expediting our detailed understanding of how coinfection with A. hydrophila and E. faecalis was controlled.

Capsaicin attenuates the effect of inflammatory cytokines in a HaCaT cell model for basal keratinocytes.

The resolution of the skin's inflammatory response is only possible if its barrier function is restored. TRPV1 channel activation plays an important role during inflammation but the effect of this activation on the skin barrier under inflammatory conditions has not been clarified. We hypothesize that it could potentially aid the keratinocyte barrier by reducing inflammatory cytokine release and promoting tight junction development. To explore the role of TRPV1 activation in inflammation, we designed and optimized an in vitro model of keratinocytes with basal epidermal layer characteristics using HaCaT cells and TNFα to induce inflammation. TNFα increased the gene expression of tight junction protein claudin 1 (CLDN1) by at least 2.60 ± 0.16-fold, in a concentration-dependent manner, over a 48h period. The administration of a capsaicin pre-treatment reduced the CLDN1 expression to 1.51 ± 0.16-fold during the first 6h after TNFα induction, whereas IL-8 cytokine release was reduced 0.64 ± 0.17-fold. After 48h, CLDN1 protein levels increased by a factor of 6.57 ± 1.39 compared to cells only treated with TNFα. These results suggest that activation of TRPV1 by capsaicin can potentiate the increase in CLDN1 expression and CLDN1 protein synthesis induced by TNFα in cultured keratinocytes, while reducing the release of IL-8.

Impaired olfactory performance and anxiety-like behavior in a rat model of multiple sclerosis are associated with enhanced adenosine signaling in the olfactory bulb via A1R, A2BR, and A3R.

The present study shows that animals with experimental autoimmune encephalomyelitis (EAE) exhibit olfactory dysfunction and impaired general cognitive abilities, as well as anxiety-like behavior. Olfactory dysfunction occurs on average at 2 dpi, well before the onset of the first motor signs of EAE (8-10 dpi). After the initial olfactory dysfunction, the EAE animals show a fluctuation in olfactory performance that resembles the relapsing-remitting course of human MS. The study also shows severe neuroinflammation in the olfactory bulb (OB), with numerous infiltrated CD4+ T cells and peripheral macrophages in the superficial OB layers, marked microgliosis, and massive induction of TNF-α, IL-1β, and IL-6. Reduced tyrosine hydroxylase activity in the glomerular layer, pronounced granule cell atrophy, and reduced numbers of type B neuroblasts in the rostral migratory stream also indicate altered plasticity of the neuronal network in the OB. Considering the exceptionally high purinome expression in the OB, the possible involvement of purinergic signaling was also investigated. The study shows that macrophages infiltrating the OB overexpress A3R, while highly reactive microglia overexpress the adenosine-producing enzyme eN/CD73 as well as A2BR, A3R, and P2X4R. Given the simultaneous induction of complement component C3, the results suggest that the microglial cells develop a functional phenotype of phagocytizing microglia. The study also demonstrates transcriptional and translational upregulation of A1R in mitral and tufted cells, which likely influence resting network activity in OB and likely contribute to olfactory dysfunction in EAE. Overall, our study shows that olfactory dysfunction and altered social and cognitive behavior in EAE are associated with increased adenosine signaling via A1R, A2BR, and A3R.

Tumor Necrosis Factor-Alpha: Ally and Enemy in Protean Cutaneous Sceneries.

Skin is the forestage for a series of many-sided functions of tumor necrosis factor-alpha (TNF-α), a proinflammatory cytokine with staggering versatility and sizable implications for tissue homeostasis, immune responses, angiogenesis, apoptosis, local and systemic inflammation. An aberrant TNF-α-mediated crosstalk has been linked to the pathogenesis of acute and chronic skin inflammatory diseases, and indeed, TNF-α dysregulation can contribute to the development and progression of psoriasis, vitiligo, local damage following exposition to ultraviolet light radiations, cutaneous lupus erythematosus, and acne vulgaris. Therapies that target TNF-α are conspicuously used in the treatment of different skin disorders, aiming to modulate the in vivo immune functions triggered by many cutaneous cells, including keratinocytes, mast cells, or Langerhans cells, and reduce inflammation taking place within the skin. Herein, we focus on the key relationships between TNF-α and distinct skin non-neoplastic inflammatory or physiologic conditions, showing that a natural induction of TNF-α may have a protective significance but that TNF-α overproduction may be harmful or even lethal. Many questions remain unraveled in the therapeutic practice, and caution should be exercised due to eventual backlashes exerted by TNF-α in maintaining skin health or in provoking skin disease.

Sex-Specific Effects of THRβ Signaling on Metabolic Responses to High Fat Diet in Mice.

Thyroid hormone (TH) plays a crucial role in regulating the functions of both bone and adipose tissue. Given that TH exerts its cholesterol-lowering effects in hepatic tissue through the TH receptor-β (TRβ), we hypothesized that TRβ agonist therapy using MGL3196 (MGL) would be effective in treating increased adiposity and bone loss in response to a 12-week high-fat diet (HFD) in adult C57BL/6J mice. Transcriptional and serum profiling revealed that HFD-induced leptin promoted weight gain in both males and females, but MGL only suppressed leptin induction and weight gain in males. In vitro studies suggest that estrogen suppresses MGL activity in adipocytes, indicating that estrogen might interfere with MGL-TRβ function. Compared to systemic adiposity, HFD reduced bone mass in male but not female mice. Paradoxically, MGL treatment reversed macroscopic bone mineral density loss in appendicular bones, but micro-CT revealed that MGL exacerbated HFD-induced trabecular bone loss, and reduced bone strength. In studies on the mechanisms for HFD effects on bone, we found that HFD induced Rankl expression in male femurs that was blocked by MGL. By ex vivo assays, we found that RANKL indirectly represses osteoblast lineage allocation of osteoprogenitors by induction of inflammatory cytokines TNFα, IL-1β, and CCL2. Finally, we found that MGL functions in both systemic adiposity and bone by nongenomic TRβ signaling, as HFD-mediated phenotypes were not rescued in TRβ147F knockout mice with normal genomic but defective nongenomic TRβ signaling. Our findings demonstrate that the negative effects of HFD on body fat and bone phenotypes are impacted by MGL in a gender-specific manner.

Non-infectious immune complexes downregulate the production of interferons and tumor necrosis factor-α in primary porcine alveolar macrophages in vitro.

Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) has been harming the pig industry worldwide for nearly 40 years. Although scientific researchers have made substantial efforts to explore PRRSV pathogenesis, the immune factors influencing PRRSV infection still need to be better understood. Infectious virus-antibody immune complexes (ICs) formed by PRRSV and sub-or non-neutralizing antibodies specific for PRRSV may significantly promote the development of PRRS by enhancing PRRSV replication through antibody-dependent enhancement. However, nothing is known about whether PRRSV infection is affected by non-infectious ICs (NICs) formed by non-pathogenic/infectious antigens and corresponding specific antibodies. Here, we found that PRRSV significantly induced the transcripts and proteins of interferon-α (IFN-α), IFN-β, IFN-γ, IFN-λ1, and tumor necrosis factor-α (TNF-α) in vitro primary porcine alveolar macrophages (PAMs) in the early stage of infection. Our results showed that NICs formed by rabbit-negative IgG (RNI) and pig anti-RNI specific IgG significantly reduced the transcripts and proteins of IFN-α, IFN-β, IFN-γ, IFN-λ1, and TNF-α in vitro PAMs and significantly elevated the transcripts and proteins of interleukine-10 (IL-10) and transforming growth factor-β1 (TGF-β1) in vitro PAMs. NICs-mediated PRRSV infection showed that NICs not only significantly decreased the induction of IFN-α, IFN-β, IFN-γ, IFN-λ1, and TNF-α by PRRSV but also significantly increased the induction of IL-10 and TGF-β1 by PRRSV and considerably enhanced PRRSV replication in vitro PAMs. Our data suggested that NICs could downregulate the production of antiviral cytokines (IFN-α/β/γ/λ1 and TNF-α) during PRRSV infection in vitro and facilitated PRRSV proliferation in its host cells by inhibiting innate antiviral immune response. This study elucidated one novel immune response to PRRSV infection, which would enhance our understanding of the pathogenesis of PRRSV.

Caspase-8 activity mediates TNFα production and restricts Coxiella burnetii replication during murine macrophage infection.

Coxiella burnetii is an obligate intracellular bacteria that causes the global zoonotic disease Q Fever. Treatment options for chronic infection are limited, and the development of novel therapeutic strategies requires a greater understanding of how C. burnetii interacts with immune signaling. Cell death responses are known to be manipulated by C. burnetii, but the role of caspase-8, a central regulator of multiple cell death pathways, has not been investigated. In this research, we studied bacterial manipulation of caspase-8 signaling and the significance of caspase-8 to C. burnetii infection, examining bacterial replication, cell death induction, and cytokine signaling. We measured caspase, RIPK, and MLKL activation in C. burnetii-infected tumor necrosis factor alpha (TNFα)/cycloheximide-treated THP-1 macrophage-like cells and TNFα/ZVAD-treated L929 cells to assess apoptosis and necroptosis signaling. Additionally, we measured C. burnetii replication, cell death, and TNFα induction over 12 days in RIPK1-kinase-dead, RIPK3-kinase-dead, or RIPK3-kinase-dead-caspase-8-/- bone marrow-derived macrophages (BMDMs) to understand the significance of caspase-8 and RIPK1/3 during infection. We found that caspase-8 is inhibited by C. burnetii, coinciding with inhibition of apoptosis and increased susceptibility to necroptosis. Furthermore, C. burnetii replication was increased in BMDMs lacking caspase-8, but not in those lacking RIPK1/3 kinase activity, corresponding with decreased TNFα production and reduced cell death. As TNFα is associated with the control of C. burnetii, this lack of a TNFα response may allow for the unchecked bacterial growth we saw in caspase-8-/- BMDMs. This research identifies and explores caspase-8 as a key regulator of C. burnetii infection, opening novel therapeutic doors.

Comparison of the transcriptome, lipidome, and c-di-GMP production between BCGΔBCG1419c and BCG, with Mincle- and Myd88-dependent induction of proinflammatory cytokines in murine macrophages

We have previously reported the transcriptomic and lipidomic profile of the first-generation, hygromycin-resistant (HygR) version of the BCGΔBCG1419c vaccine candidate, under biofilm conditions. We recently constructed and characterized the efficacy, safety, whole genome sequence, and proteomic profile of a second-generation version of BCGΔBCG1419c, a strain lacking the BCG1419c gene and devoid of antibiotic markers. Here, we compared the antibiotic-less BCGΔBCG1419c with BCG. We assessed their colonial and ultrastructural morphology, biofilm, c-di-GMP production in vitro, as well as their transcriptomic and lipidomic profiles, including their capacity to activate macrophages via Mincle and Myd88. Our results show that BCGΔBCG1419c colonial and ultrastructural morphology, c-di-GMP, and biofilm production differed from parental BCG, whereas we found no significant changes in its lipidomic profile either in biofilm or planktonic growth conditions. Transcriptomic profiling suggests changes in BCGΔBCG1419c cell wall and showed reduced transcription of some members of the DosR, MtrA, and ArgR regulons. Finally, induction of TNF-α, IL-6 or G-CSF by bone-marrow derived macrophages infected with either BCGΔBCG1419c or BCG required Mincle and Myd88. Our results confirm that some differences already found to occur in HygR BCGΔBCG1419c compared with BCG are maintained in the antibiotic-less version of this vaccine candidate except changes in production of PDIM. Comparison with previous characterizations conducted by OMICs show that some differences observed in BCGΔBCG1419c compared with BCG are maintained whereas others are dependent on the growth condition employed to culture them.

Induction of TNFα by flavin adenine dinucleotide (FADH) protects neonatal C57Bl6 lungs from high oxygen induced lung injury

Background: Bronchopulmonary dysplasia (BPD) is a serious lung disease caused by oxidative stress, predominately affecting premature infants that require prolonged oxygen support. The co-factor flavin adenine dinucleotide (FADH) facilitates glutathione reductase (GR) enzymatic activity and increases the bioavailability of the antioxidant glutathione (GSH). As such, we hypothesized that intranasal delivery of FADH can improve redox homeostasis and attenuate lung injury following chronic high oxygen supplementation (0.85% FiO2) by altering pro-inflammatory signal transduction pathways in a C57Bl6 newborn mouse model for BPD. Methods: We used a prolonged hyperoxia-induced lung injury rodent model to recapitulate the clinical and histological changes present in BPD. Newborn mice were housed in FiO2 85% (hyperoxic) or 21% O2 (normoxic) from day of birth until post-natal day (PN) 14. FADH (7 μM) or saline (control) treatments were administered daily via intranasal insufflation on PN 14-21 in a final volume of 1.5μL/g body weight. BALF and lung tissue samples were collected and evaluated for redox stress, lung injury/development, and inflammation using standard immunohistological techniques, morphometric analysis, and standard molecular assays. BALF cytokines were measured using a flow cytometric approach (BioLegend). Two-dimensional cell migration assays were tracked via microscopy daily until 100% confluence reached; the ½ maximal peak values were evaluated to compare rate of wound closure following very low (2.5 ng/mL) TNFα or IL12 treatment. Transepithelial resistance and potential differences were measured using an EVOM device (World Precision Instrument) across human small airway epithelial cells, and equivalent short circuit currents (Isc) calculated in accordance with Ohm’s Law, where V=IR. Single data comparisons were performed using paired Student’s t-tests. Multiple comparisons were performed using ANOVA followed by post-hoc testing in Sigma Plot. Results: FADH protected neonatal lungs from high oxygen induced oxidative stress: GSH/GSSG Eh (a measure of oxidative stress) improved from -168.77 mV ± 3.64 mV to -179.10 mV ± 1.85 mV (n=5 BALF measurements). FADH also improved lung injury scores from 0.187±0.038 to 0.03±0.014 (p=0.005), decreased neutrophil migration (p<0.001), and increased macrophages (p<0.001) when compared to age-matched untreated pups. FADH-mediated increase in TNFα (and not IL12) significantly increased the rate of lung epithelial cell wound closure from [2.27±0.07 to 1.97±0.1 average days for ½ complete wound closure, n=6, p=0.01] and increased Isc from 6.5±.21 to 9.2±1.0 μA/cm2, n=3, p=0.03. Conclusions: Our findings indicate that FADH protects the preterm lung from hyperoxic injury by 1) increasing the bioavailability of GSH and 2) via TNFα signaling to increase the rate of wound closure and repair of epithelial cell function. Significances: The mechanisms identified through this study could be targeted as a potential adjunct therapy for BPD patients to attenuate high oxygen induced lung injury. Funding: Supported by R01HL137033 (MH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Elevated Kallistatin promotes the occurrence and progression of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, and the development of non-alcoholic steatohepatitis (NASH) might cause irreversible hepatic damage. Hyperlipidemia (HLP) is the leading risk factor for NAFLD. This study aims to illuminate the causative contributor and potential mechanism of Kallistatin (KAL) mediating HLP to NAFLD. 221 healthy control and 253 HLP subjects, 62 healthy control and 44 NAFLD subjects were enrolled. The plasma KAL was significantly elevated in HLP subjects, especially in hypertriglyceridemia (HTG) subjects, and positively correlated with liver injury. Further, KAL levels of NAFLD patients were significantly up-regulated. KAL transgenic mice induced hepatic steatosis, inflammation, and fibrosis with time and accelerated inflammation development in high-fat diet (HFD) mice. In contrast, KAL knockout ameliorated steatosis and inflammation in high-fructose diet (HFruD) and methionine and choline-deficient (MCD) diet-induced NAFLD rats. Mechanistically, KAL induced hepatic steatosis and NASH by down-regulating adipose triglyceride lipase (ATGL) and comparative gene identification 58 (CGI-58) by LRP6/Gɑs/PKA/GSK3β pathway through down-regulating peroxisome proliferator-activated receptor γ (PPARγ) and up-regulating kruppel-like factor four (KLF4), respectively. CGI-58 is bound to NF-κB p65 in the cytoplasm, and diminishing CGI-58 facilitated p65 nuclear translocation and TNFα induction. Meanwhile, hepatic CGI-58-overexpress reverses NASH in KAL transgenic mice. Further, free fatty acids up-regulated KAL against thyroid hormone in hepatocytes. Moreover, Fenofibrate, one triglyceride-lowering drug, could reverse hepatic steatosis by down-regulating KAL. These results demonstrate that elevated KAL plays a crucial role in the development of HLP to NAFLD and may be served as a potential preventive and therapeutic target.

ATP-P2X7 signaling mediates brain pathology while contributing to viral control in perinatal Zika virus infection

Zika virus (ZIKV), the causative agent of Zika fever, is a flavivirus transmitted by mosquitoes of the Aedes genus. Zika virus infection has become an international concern due to its association with severe neurological complications such as fetal microcephaly. Viral infection can induce the release of ATP in the extracellular environment, activating receptors sensitized by extracellular nucleotides, such as the P2X7 receptor. This receptor is the primary purinergic receptor involved in neuroinflammation, neurodegeneration, and immunity. In this work, we investigated the role of ATP-P2X7 receptor signaling in Zika-related brain abnormalities. Wild-type mice (WT) and P2X7 receptor-deficient (P2X7-/-) C57BL/6 newborn mice were subcutaneously inoculated with 5 × 106plaque-forming units of ZIKV or mock solution. P2X7 receptor expression increased in the brain of Zika virus-infected mice compared to the mock group. Comparative analyses of the hippocampi from WT and P2X7-/-mice revealed that the P2X7 receptor increased hippocampal damage in CA1/CA2 and CA3 regions. Doublecortin expression decreased significantly in the brains of ZIKV-infected mice. WT ZIKV-infected mice showed impaired motor performance compared to P2X7-/- infected mice. WT ZIKV-infected animals showed increased expression of glial markers GFAP (astrocytes) and IBA-1 (microglia) compared to P2X7-/- infected mice. Although the P2X7 receptor contributes to neuronal loss and neuroinflammation, WT mice were more efficient in controlling the viral load in the brain than P2X7 receptor-deficient mice. This result was associated with higher induction of TNF-α, IFN-β, and increased interferon-stimulated gene expression in WT mice than P2X7-/-ZIKV-infected. Finally, we found that the P2X7 receptor contributes to inhibiting the neuroprotective signaling pathway AKT/mTOR while stimulating the caspase-3 activation, possibly two distinct pathways contributing to neurodegeneration. These findings suggest that ATP-P2X7 receptor signaling contributes to the antiviral response in the brain of ZIKV-infected mice while increasing neuronal loss, neuroinflammation, and related brain abnormalities.

Loss of Endothelial Annexin A1 Aggravates Inflammation-Induched Vascular Aging.

Chronic inflammation is increasingly considered as the most important component of vascular aging, contributing to the progression of age-related cardiovascular diseases. To delay the process of vascular aging, anti-inflammation may be an effective measure. The anti-inflammatory factor annexin A1 (ANXA1) is shown to participate in several age-related diseases; however, its function during vascular aging remains unclear. Here, an ANXA1 knockout (ANXA1-/-) and an endothelial cell-specific ANXA1 deletionmouse (ANXA1△EC) model are used to investigate the role of ANXA1 in vascular aging. ANXA1 depletion exacerbates vascular remodeling and dysfunction while upregulatesage- and inflammation-related protein expression. Conversely,Ac2-26 (a mimetic peptide of ANXA1) supplementation reverses this phenomenon. Furthermore, long-term tumor necrosis factor-alpha (TNF-α) induction of human umbilical vein endothelial cells (HUVECs) increases cell senescence. Finally, the senescence-associated secretory phenotype and senescence-related protein expression, rates of senescence-β-galactosidasepositivity, cell cycle arrest, cell migration, and tube formation ability are observed in both ANXA1-knockdown HUVECs and overexpressed ANXA1-TNF-α induced senescent HUVECs. They also explore the impact of formylpeptide receptor 2 (a receptor of ANXA1) in an ANXA1 overexpression inflammatory model. These data provide compelling evidence that age-related inflammation in arteries contributes to senescent endothelial cells that promote vascular aging.

Hinokitiol as a modulator of TLR4 signaling and apoptotic pathways in atopic dermatitis

Atopic dermatitis (AD) poses a significant global health challenge, characterized by dysregulated inflammation and apoptotic processes. This study explores the therapeutic efficacy of hinokitiol, employing a comprehensive in vivo and in vitro approach. Assessment of inflammation-related markers in the animal model included observation of physical appearance, Western blotting, ELISA, and H&E staining. Additionally, the cell culture model enabled the evaluation of apoptosis and ROS levels using MTT assay, crystal violet staining, Western blot, and DCFDA assays. The results revealed hinokitiol's proficiency in ameliorating ear and skin morphology in the DNCB-induced AD model, mediated through the TLR4/MyD88 pathway. Notably, hinokitiol intervention led to a reduction in both M1 and M2 macrophage phenotypes. In vitro investigations demonstrated hinokitiol's ability to enhance cell viability and morphology under TNF-α and IFN-γ induction. Mechanistically, hinokitiol exhibited regulatory effects on apoptosis-related proteins, including Bax, Cytochrome c, Caspase-3, and PARP, thereby averting cellular damage. These findings suggest that hinokitiol is a promising natural compound with significant potential for alleviating inflammation and apoptosis in AD, indicating potential avenues for future therapeutic developments.

Molecular Determinants of Response to Mosunetuzumab Plus CHOP in Patients with Previously Untreated (1L) Diffuse Large B Cell Lymphoma (DLBCL)

Co-lead authors: Habib Hamidi and Patrick Kimes Background: Standard induction therapy cures ~60% of patients (pts) with DLBCL; however, most remaining non-responders die from lymphoma, highlighting a need for improved first-line treatments. Biomarkers that can reliably assess the efficacy of new treatments are lacking in clinical practice. Mosunetuzumab (Mosun) is a CD20xCD3 T cell engaging bispecific antibody that redirects T cells to eliminate malignant B cells. In a Phase Ib/II trial (NCT03677141), Mosun combined with CHOP chemotherapy (M-CHOP) induced high response rates in pts with 1L DLBCL (ORR 87.5%, CR 85%; Phillips et al. ASH 2020). Here, we sought to identify biomarkers associated with M-CHOP response. Methods: Pts with 1L DLBCL receiving M-CHOP (six cycles) in NCT03677141 and with available biomarker data were included. Bulk RNASeq data from pre-treatment tumor biopsies were analyzed using gene set variation for tumor intrinsic pathways and xCell cell-type enrichment for immune and stromal cell types. High or low expression of each gene signature was determined by median cut-off. Cox regression examined associations between gene signatures and PFS. To assess specificity to M-CHOP, gene signatures were examined in pre-treatment biopsies from pts treated with rituximab + CHOP chemotherapy (R-CHOP) in GOYA (NCT01287741; Sehn et al. J Hematol Oncol 2020). Peripheral blood biomarkers were evaluated by whole blood flow cytometry and plasma cytokines by ELISA. Circulating tumor (ct) DNA levels were measured at baseline and on treatment using the AVENIO NHL CAPP-Seq assay (Stokowski et al. ASH 2022). Results: In M-CHOP-treated pts (n=33), gene expression analysis revealed two types of signatures associated with PFS: tumor immune microenvironment signature (TIMS) and tumor intrinsic proliferation signature (TIPS; Figure). High TIMS expression (e.g., CD4+ T cells) was associated with significantly longer PFS than low expression. Conversely, pts with high TIPS expression (e.g., MYC targets, DNA repair) had shorter PFS than those with low expression. These biological associations were specific to M-CHOP and were not observed in R-CHOP-treated pts. Consistent with the tumor-gene expression analysis, immune profiling of peripheral blood suggested that in pts treated with M-CHOP, a higher T cell baseline count was associated with a trend for prolonged PFS compared with pts with a lower baseline count. A higher count of circulating suppressive T cells was also associated with shortened PFS. Early on-treatment pharmacodynamic (PD) changes were observed following the first Mosun dose, including: induction of TNFα and IL-6, transient margination of CD4+ and CD8+ T cells, and activation of CD4+ and CD8+ T cells. The magnitude of the PD effects were similar to observations with Mosun monotherapy in pts with relapsed or refractory DLBCL (Hernandez et al. ASH 2019), confirming that Mosun maintains its immune activation effect when combined with CHOP in pts with 1L DLBCL. High baseline ctDNA levels were associated with high IPI score (n=32, Wilcoxon rank-sum p=0.007), ABC/non-GCB DLBCL subtype (n=30, p=0.033), and high lactate dehydrogenase (n=32, p=0.004). Pts with baseline ctDNA levels < median had a modest trend for improved PFS compared with pts > median (unadjusted hazard ratio [HR]=0.57 [95% CI 0.16, 1.75]; IPI-adjusted HR=0.72 [95% CI 0.16, 3.20]). The proportion of pts with undetectable ctDNA increased with each M-CHOP cycle (C) (C2 day [D]1, 25% [8/32]; C3D1, 69% [20/29]; C5D1, 88% [22/25]). Pts with undetectable ctDNA at any on-treatment time point (C2D1, C3D1, C5D1) had superior PFS compared with pts with detectable ctDNA (unadjusted HR=0.12 [95% CI 0.03, 0.52]; IPI adjusted HR=0.14 [95% CI 0.03, 0.61]). Conclusions: Consistent with the Mosun mode of action, pts with high expression of TIMS and those with high levels of circulating T cells had improved PFS versus pts with low expression. In contrast, there was no association between immune cell signatures and PFS in pts treated with R-CHOP, suggesting that the different mode of action of rituximab versus Mosun may influence this. Future studies are warranted to confirm the potential clinical utility of immune cell signatures as biomarkers associated with CD20xCD3 bispecifics' activity.

PRRSV-Vaccinated, Seronegative Sows and Maternally Derived Antibodies (I): Impact on PRRSV-1 Challenge Outcomes in Piglets.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) remains an infectious agent with high importance in the swine industry. In this study, the influence of maternally derived antibodies (MDAs) on an experimental PRRSV-1 challenge is investigated. Piglets included in the study (n = 36) originated from a Belgian farrow-to-finish herd in which the sow population was routinely vaccinated with a modified live vaccine against PRRSV. Eighteen piglets were born from three PRRSV-seropositive sows (responders to vaccination) and had a clear presence of PRRSV-specific MDAs (E+ piglets). The other eighteen piglets were born from three PRRSV-seronegative sows (non-responders to vaccination) and did not have PRRSV-specific MDAs (E- piglets). In each group, twelve piglets were intranasally challenged with a high dose of the heterologous PRRSV-1 07V063 strain, the remaining piglets were mock-challenged (PBS) and served as controls. During the first days after infection, higher serum viremia and nasal shedding were observed in the challenged E- piglets compared to the challenged E+ piglets. However, at 10 days post-infection, the peak serum viremia was significantly higher in the E+ piglets in comparison to the E- piglets and serum viremia remained slightly higher in this group until the end of the study. Additionally, the two challenged groups had a different immune response to the PRRSV infection. The E- challenged piglets showed an earlier and more intense seroconversion, leading to significantly higher antibody titers at 10 dpi compared to the E+ challenged piglets. Furthermore, a trend towards both higher induction of serum IFN-γ and higher induction of IFN-γ secreting cells was observed in the E- challenged piglets. In contrast, a significantly higher induction of serum TNF-α at 7 dpi was seen in the E+ challenged piglets compared to the E- challenged piglets. The results gathered in this study suggest that PRRSV-specific MDAs induce partial protection during the early stages of infection but are not sufficient to protect against a high challenge dose. The presence of piglets lacking PRRSV-specific MDAs might pose a risk for PRRSV infection and enhanced transmission in pig farms in young piglets.

Revisiting Coley's Toxins: Immunogenic Cardiolipins from Streptococcus pyogenes.

Coley's toxins, an early and enigmatic form of cancer (immuno)therapy, were based on preparations of Streptococcus pyogenes. As part of a program to explore bacterial metabolites with immunomodulatory potential, S. pyogenes metabolites were assayed in a cell-based immune assay, and a single membrane lipid, 18:1/18:0/18:1/18:0 cardiolipin, was identified. Its activity was profiled in additional cellular assays, which showed it to be an agonist of a TLR2-TLR1 signaling pathway with a 6 μM EC50 and robust TNF-α induction. A synthetic analog with switched acyl chains had no measurable activity in immune assays. The identification of a single immunogenic cardiolipin with a restricted structure-activity profile has implications for immune regulation, cancer immunotherapy, and poststreptococcal autoimmune diseases.

Induction of TNF-α by Filifactor alocis in THP-1 macrophagic cells

ObjectivesFilifactor alocis is an emerging periodontal pathogen, and macrophage-produced tumor necrosis factor-α (TNF-α) plays important roles in periodontal pathogenesis. In this study, we investigated F. alocis-stimulated TNF-α production in THP-1 macrophagic cells. DesignPhorbol 12-myristate 13-acetate-differentiated THP-1 macrophagic cells were challenged with F. alocis ATCC 35896 for various durations. TNF-α mRNA expression and protein secretion were determined using RT-PCR and ELISA, respectively. Activation of protein kinases and transcription factor proteins was evaluated by Western blot analysis. ResultsLive F. alocis stimulated THP-1 cells to produce TNF-α in a dose-dependent manner. However, glutaraldehyde-killed or heat-killed F. alocis showed no effectiveness for TNF-α induction. In contrast, both live and killed Porphyromonas gingivalis robustly increased TNF-α expression. Furthermore, F. alocis was unable to stimulate TNF-α expression in Toll-like receptor 2 (TLR2) knockout THP-1 cells. F. alocis activated all three mitogen-activated protein kinases: extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Pharmacological inhibition of ERK and JNK, but not p38, significantly reduced F. alocis-induced TNF-α production. Finally, increased levels of phospho-c-Jun were detected in F. alocis-stimulated THP-1 cells. ConclusionsThese results suggest that F. alocis induces TNF-α production in THP-1 macrophagic cells primarily by activating the TLR2, JNK, and c-Jun pathways.

TNF-α and Poly(I:C) induction of A20 and activation of NF-κB signaling are independent of ABCF1 in human airway epithelial cells

ABCF1 is the most characterized member of the ABCF family in eukaryotes with proposed functions related to innate immunity in fibroblasts, macrophages, and epithelial cells. Currently, a mechanistic link between ABCF1 and immune responses in human airway epithelial cells (HAECs) remains to be clearly defined. The present study aimed at characterizing the function of ABCF1 in the context of nuclear factor nuclear factor κB (NF-κB) mediated pro-inflammatory responses in an immortalized human airway epithelial cell line, HBEC-6KT. We demonstrated that with ABCF1 silencing under basal conditions, TNF Alpha Induced Protein 3 (TNFAIP3/A20) protein expression and downstream expression and activation of transcription factors, NF-κB and Interferon regulatory factor 3 (IRF-3), were not disrupted. We followed with investigations of ABCF1 function under a pro-inflammatory stimuli that are known to be regulated by A20. We demonstrated that under Polyinosinic:polycytidylic acid (Poly(I:C)) and tumor Necrosis Factor-α (TNF-α) challenge with ABCF1 silencing, there was a significant reduction in secreted levels of interleukin-8 (IL-8) and a trend for reduced IL-6. However, we observed no changes to the expression levels of A20 and the activation status of the transcription factors, NF-κB and IRF-3. Collectively, these studies demonstrate that Poly(I:C) and TNF-α induced IL-8 is regulated by ABCF1 via pathways independent of NF-κB and IRF-3 activation.

Abstract P416: Differences In Immune Cell Composition And Response Between Spontaneously Hypertensive Bph/2 Mice And Normotensive Bpn/3 Mice

There is considerable evidence for a role for the immune system in different animal models of hypertension. However, whether there are differences in the immune systems of Bph/2 spontaneously hypertensive mice and Bpn/3 normotensive mice is not known. To address this, we characterized the immune composition and response of male Bph/2 and Bpn/3 mice. Bph/2 mice had significantly lower body weight compared to Bpn/3 mice (Bpn: 32.0 ± 0.5 g; Bph: 23.0 ± 0.5 g) with a proportionately lower spleen weight (Bpn: 70.1 ± 9.8 mg; Bph: 48.6 ± 2.1 mg). As expected, Bph/2 mice had higher blood pressure than Bpn/3 mice. Bph/2 mice also had a higher percentage of CD4 T cells in brachial lymph nodes compared to Bpn/3 mice (Bpn: 48.0 ± 0.57, Bph: 58.6 ± 1.94) and a lower percentage of CD8 T cells (Bpn: 46.4 ± 1.07, Bph: 38.9 ± 1.84), causing an increased CD4:CD8 T cell ratio in Bph/2 mice (Bpn: 1.04 ± 0.03, Bph: 1.53 ± 0.12). This trend was also observed in inguinal lymph nodes, but not in spleens or mesenteric lymph nodes. There were also differences in the response of anti-CD3/anti-CD28-activated splenocytes. Specifically, IFNγ secretion was markedly lower in splenic T cells from Bph/2 mice compared to those from Bpn/3 mice 24 h after activation (Bpn: 4902 ± 1532 pg/ml, Bph/2: 921 ± 462 pg/ml). Similarly, IL-2 (Bpn: 3960 ± 1070 pg/ml; Bph: 1121 ± 247 pg/ml), IL-6 (Bpn: 297 ± 94 pg/ml, Bph: 64 ± 15 pg/ml) and TNFα (Bpn: 170 ± 33 pg/ml; Bph: 59 ± 15 pg/ml) induction was also lower in T cells from Bph/2 mice. Interestingly, we also found that Bpn/3 mice do not express the NK cell marker, NK1.1. Taken together, the data suggest significant differences in immune composition and response between Bph/2 and Bpn/3 mice and indicate a need to further evaluate the role of the immune system in the development of hypertension in this model.