Journal of Immunotoxicology最新文献

Macrophage polarization has been demonstrated to exert a vital role on asthma pathogenesis. Mesenchymal stem cells (MSC) have the capacity to modulate macrophage differentiation from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 phenotype. However, the impact of MSC-macrophage interactions on asthma development and underlying mechanisms responsible for this interaction remain largely unknown. The aim of this study was to investigate the role of AhR expressed on MSC in macrophage polarization in a cockroach extract (CRE)-induced asthma mouse model. The studies here revealed that MSC polarized macrophages from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 phenotype in this model. The mRNA levels of interleukin (IL)-6, IL-1β, and NOS2 as M1 markers were significantly decreased while those of select M2 markers such as Arg-1, FIZZ1, and YM-1 were significantly enhanced. It was also observed that aryl hydrocarbon receptor (AhR) signaling was significantly increased during asthma pathogenesis as demonstrated by enhanced mRNA expression of AhR, CYP1a1, and CYP1b1. It was also seen that the elevated AhR signaling was able to attenuate the onset of asthma. Use of an AhR antagonist (CH223191) resulted in significant inhibition of the AhR signaling and increases in M2 marker expression, but led to elevation of expression of M1 markers in the CRE-induced asthma model. Taken together, the current study showed that MSC can modulate macrophage polarization, in part, via activation of AhR signaling during CRE-induced asthma.

Exposure to environmental toxicants that affect the immune system and overall health of many mammals is mostly unavoidable. One of the more common substances is the mycotoxins, especially carcinogenic aflatoxin (AF)B₁ which also causes immune suppression/dysregulation in exposed hosts. The present study analyzed the effects of naturally occurring levels of AFB₁ on apoptosis of healthy bovine and camelid neonatal neutrophils (PMN) that were isolated both before and after host consumption of colostrum. Cells from bovine and camel neonates (n = 12 sets of PMN/mammal/timepoint) were exposed for 24 h to a low level of AFB₁ (i.e. 10 ng AFB₁/ml) and then intracellular ATP content and caspase-3, -7, and -9 activities (determined by bioluminescence) were assessed. The results indicated a significant lessening of intracellular ATP content and equivalents of luminescence intensity in AFB₁-treated PMN in all studied samples, i.e. isolated pre-and post-colostrum consumption. In contrast, caspase-3, -7, and -9 activities in both pre- and post-colostrum consumption bovine and camelid PMN were noticeably increased (∼>2-fold). The damaging effects of AFB₁ were more pronounced in bovine neonate PMN than in camelid ones. These results showed that camelid or bovine neonatal PMN collected pre- and post-colostrum are sensitive (moreso after consumption) to naturally occurring levels of AFB₁. While merits of colostrum are well known, its failure to mitigate toxic effects of AFB₁ in what would translate into a critical period in the development of immune competence (i.e. during the first few days of life in bovine and camelid calves) is surprising. The observed in vitro toxicities can help clarify underlying mechanisms of immune disorders caused by AFs in animals/humans.

Mast cells play key roles in allergy, anaphylaxis/anaphylactoid reactions, and defense against pathogens/toxins. These cells contain cytoplasmic granules with a wide spectrum of pleotropic mediators that are released upon activation. While mast cell degranulation (MCD) occurs upon clustering of the IgE receptor bound to IgE and antigen, MCD is also triggered through non-IgE-mediated mechanisms, one of which is via Mas-related G protein-coupled receptor X2 (MRGPRX2). MRGPRX2 can be activated by many basic biogenic amines and peptides. Consequently, MRGPRX2-mediated MCD is an important potential safety liability for peptide therapeutics. To facilitate peptide screening for this liability in early preclinical drug development, a rapid, high-throughput engineered CHO-K1 cell-based MRGPRX2 activation assay was evaluated and compared to histamine release in CD34⁺ stem cell-derived mature human mast cells as a reference assay, using 30 positive control and 29 negative control peptides for MCD. Both G protein-dependent (Ca²⁺ endpoint) and -independent (β-arrestin endpoint) pathways were assessed in the MRGPRX2 activation assay. The MRGPRX2 activation assay had a sensitivity of 100% for both Ca²⁺ and β-arrestin endpoints and a specificity of 93% (β-arrestin endpoint) and 83% (Ca²⁺ endpoint) compared to histamine release in CD34⁺ stem cell-derived mature human mast cells. These findings suggest that assessing MRGPRX2 activation in an engineered cell model can provide value as a rapid, high-throughput, economical mechanism-based screening tool for early MCD hazard identification during preclinical safety evaluation of peptide-based therapeutics.

The prevalence of iron (Fe) deficiency and subclinical lead (Pb) toxicity is high in developing countries like India, and information on their potential additive effects on immune responses is scant. The current study assessed immune parameters in dual Pb-exposedFe-deficient weanling SD rats. Rats were fed a control (CD) or Fe-deficient (ID) diet for 4 weeks and then evaluated for hemoglobin (Hb) and serum Fe status. Then, half the rats in each group began to receive daily oral Pb exposure (25 mg/4 ml/kg BW; gavage) or vehicle for a further 4 weeks (while maintained on original respective diets). After the 4-weeks of dosing, rats were assessed for Hb and serum Fe, and for blood lead level (BLL) and δ-aminolevulinic acid dehydratase (ALAD) activity. At this point, half the rats in each group (now n = 8) were then vaccinated with tetanus toxoid (TT), and then two boosters at 2-week intervals. All the time, rats stayed on their original respective diets along with exposure to Pb on alternate days. At 2 weeks after the final booster, rats were euthanized and blood collected to assess total/specific IgG and IgM levels; mucosal (intestinal) IgA levels were also determined. Spleens were taken to assess CD4⁺ and CD8⁺ cell levels and for ex vivo measures of splenocyte proliferation/T_H1 and T_H2 cytokine formation. The results indicated significant lowering of Hb and serum Fe levels in ID rats and increased blood Pb and decreased ALAD activity in all Pb-exposed rats. Fe-deficiency alone induced significant increases in ALAD activity, but only in an absence of Pb. While there was no impact of any regimen on total or TT-specific IgG, significant decreases in mucosal IgA and TT-specific IgM were seen in ID-fed Pb-exposed rats. CD4⁺ cell levels were not impacted by treatment; CD8⁺ levels were increased in all ID/Pb-exposed rats. Ex-vivo splenocyte proliferation was significantly higher among vaccinated rats, as well as ID-fed Pb-exposed unvaccinated rats. Cytokine formation in all cases was highly variable. The results suggest that Fe deficiency compromised cell-mediated, mucosal, and/or humoral immune response-related endpoints and that Pb exposure during the deficiency further impacted these outcomes.

Many persistent organic pollutants, such as polychlorinated biphenyls (PCBs), have high immunomodulating potentials. Exposure to them, in combination with virus infections, has been shown to aggravate outcomes of the infection, leading to increased viral titers and host mortality. Expression of immune-related microRNA (miR) signaling pathways (by host and/or virus) have been shown to be important in determining these outcomes; there is some evidence to suggest pollutants can cause dysregulation of miRNAs. It was thus hypothesized here that modulation of miRNAs (and associated cytokine genes) by pollutants exerts negative effects during viral infections. To test this, an in vitro study on chicken embryo fibroblasts (CEF) exposed to a PCB mixture (Aroclor 1260) and then stimulated with a synthetic RNA virus (poly(I:C)) or infected with a lymphoma-causing DNA virus (Gallid Herpes Virus 2 [GaHV-2]) was conducted. Using quantitative real-time PCR, expression patterns for mir-155, pro-inflammatory TNFα and IL-8, transcription factor NF-κB1, and anti-inflammatory IL-4 were investigated 8, 12, and 18 h after virus activation. The study showed that Aroclor1260 modulated mir-155 expression, such that a down-regulation of mir-155 in poly(I:C)-treated CEF was seen up to 12 h. Aroclor1260 exposure also increased the mRNA expression of pro-inflammatory genes after 8 h in poly(I:C)-treated cells, but levels in GaHV-2-infected cells were unaffected. In contrast to with Aroclor1260/poly(I:C), Aroclor1260/GaHV-2-infected cells displayed an increase in mir-155 levels after 12 h compared to levels seen with either individual treatment. While after 12 h expression of most evaluated genes was down-regulated (independent of treatment regimen), by 18 h, up-regulation was evident again. In conclusion, this study added evidence that mir-155 signaling represents a sensitive pathway to chemically-induced immunomodulation and indicated that PCBs can modulate highly-regulated innate immune system signaling pathways important in determining host immune response outcomes during viral infections.

Cadmium (Cd) is accumulated in human astrocytes and induces the production of interleukin (IL)-6 and IL-8. Astrocytes are one of the major sources of chemokine C-C motif ligand 2 (CCL2; known as monocyte chemoattractant protein-1 [MCP-1]), in the brain. Elevated CCL2 levels are associated with cognitive impairment as well as the migration and invasion of glioblastoma cells. The present study hypothesized that non-toxic concentrations of Cd (as cadmium chloride [CdCl₂]) could up-regulate CCL2 production in U-87 MG human glio-blastoma cells. The results showed that after exposure of the U-87 MG cells to CdCl₂ at 1 and 10 µM, there was an up-regulation of CCL2 mRNA expression after 3 h of exposure and increased CCL2 secretion after 6 and 24 h. The study also found that inhibition of MAPK pathways, including ERK1/2, p38, and JNK by U0126, SB203580 and SP600125, respectively, reduced Cd-induced CCL2 secretion by the cells. Moreover, when cells were pretreated with Ro 32-0432 (an inhibitor of calcium-dependent PKC) and LY294002 (a PI3K inhibitor), this also resulted in a down-regulation of any Cd-induced CCL2 expression. Taken together, the results of this study allow for the conclusion to be made that CCL2 up-regulation in U-87 MG cells induced by Cd is mediated, in part, by an activation of MAPK, PI3K/Akt, and PKC pathways.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint which may be engaged by cells in a tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda^®) is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG₄/κ isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. The current work was focused on developing a mouse T-Dependent Antibody Response (TDAR) model using a murinized rat anti-mouse PD-1 antibody (muDX400; a rodent surrogate for pembrolizumab) to evaluate the potential impact of treatment with a PD-1 inhibitor on immune responses to an antigen challenge (e.g. HBsAg in Hepatitis B vaccine). Despite the lower binding affinity and T_1/2 compared to pembrolizumab, ligand blocking data indicated muDX400 had appropriate pharmacological activity and demonstrated efficacy in mouse tumor models, thus was suitable for pharmacodynamic and vaccination studies in mice. In a vaccination study in which mice were concomitantly administered muDX400 and the Hepatitis B vaccine, muDX400 was well-tolerated and did not result in any immune-mediated adverse effects. The treatment with muDX400 was associated with a shift in the ratio between naive and memory cells in both CD4⁺ and CD8⁺ T-lymphocytes in the spleen but did not affect anti-HBsAg antibody response profile. The mouse TDAR model using the Hepatitis B vaccine and the surrogate anti-PD1 monoclonal antibody was a useful tool in the evaluation of the potential immune-mediated effects of pembrolizumab following vaccination and appears to be a suitable alternative for the nonhuman primate TDAR models utilized for other checkpoint inhibitors.

There is increasing evidence that host inflammatory responses play an important role in the development and progression of cancers. There are some data that cancer is associated not only with inflammation at the site of the lesion, but also with dysregulations of the host overall systemic immune response. In the case of cervical cancer, inflammation is an important factor associated with the development, progression, and potential metastasis of the disease. What is unclear still in the potential for modifications of host responses to human papillomaviruses (HPV) - a known causative agent of CC, that could be induced by cigarette smoking. In particular, it remains to be determined how the inflammation induced by HPV infection could impact on CC incidence/severity. In this prospective study, serum levels of 10 cytokines were evaluated using Multiplex and ELISA assays. The samples were the sera of 43 CC patients and 60 healthy (NILM) controls. All outcomes were evaluated in relation to host HPV and to their smoking status. The results in indicated that serum sTREM-1, TNFα, IFNβ, IL-1β, and IL-6 levels were significantly increased in CC (HPV+) patients compared to healthy NILM controls. A similar trend was observed for IL-10 and IL-2 levels. Within the two groups, differences in cytokine levels between smokers and never smokers were not remarkable. The findings here support the hypothesized role of systemic inflammation in the pathophysiology of CC.

Currently, there is a multitude of CD3 bispecifics with different molecular designs and binding properties in preclinical and clinical development for the treatment of liquid or solid tumors. The key safety concerns with CD3 bispecifics are excessive release of cytokines, which may translate to potentially life-threating cytokine release syndrome (CRS), target organ toxicity due to redirection of T-cells to normal tissues expressing the tumor-associated antigen (TAA) (off-tumor/on-target cytotoxicity), and, in some instances, neurotoxicity. Another key challenge is to arrive at a safe clinical starting dose and an efficient escalating strategy that allows patients in early dose cohorts the potential for clinical benefit in Phase 1 trials. To expand the therapeutic index and bring more treatment options to patients, there are intense efforts to overcome these challenges through improvements in molecular design, preclinical safety assessment strategies, and clinical management practices. A recent workshop at the U.S. Food and Drug Administration (FDA) with industry, academic, and regulatory agency representation was held to discuss the challenges and explore where such improvements to the development of CD3 bispecifics can be implemented. Here, the content of the presentations and the discussion that occurred during this workshop are summarized.

In the study here, the potential applicability of KMRC011 - an agonist of toll-like receptor-5 - as a countermeasure for radiation toxicities was evaluated. Following a single 5.5 Gy total body irradiation (TBI, surface absorbed dose = 7 Gy) of Co⁶⁰ γ-rays, mortality rates and degrees of pathological lesions that developed over 80 days were compared in monkeys that received TBI only and a group that was injected once with KMRC011 (10 μg/kg) after TBI. Compared to the TBI-only hosts (80%), the death rate was significantly improved by the use of KMRC011 (40%), all deaths in both groups occurred in the period from Days 19-24 post-TBI. Further analysis of monkeys that survived until the end of the experiment showed that AST and ALT levels were elevated only in the TBI group, and that radiation-induced tissue damage was alleviated by the KMRC011 injection. Additionally, expression of cell death-related proteins was lower in tissues from the KMRC011-treated hosts than in those in the TBI-only group. Other measured parameters, including body weight, food uptake, and hematological values did not significantly differ between the two groups over the entire period. The results of this study, thus demonstrate that KMRC011 could potentially be used as a medical countermeasure for the treatment of acute radiation exposure.