Preliminary Evidence of Endotoxin Tolerance in Dairy Cows during the Transition Period

The blastogenic response of bovine peripheral blood mononuclear cells (PBMCs) to lipopolysaccharides (LPS) has been investigated for a long time in our laboratories. In particular, a possible correlation between the blastogenic response to LPS and the disease resistance of dairy cows has been suggested in previous studies. Isolated PBMCs from eight cows at three different time points during the transition period (T0 = 15 days before calving; T1 = 7 days post-calving; T2 = 21 days post-calving) were cultured in the presence or absence of LPS, and the blastogenic response was assayed 72 h after in vitro stimulation. Moreover, the gene expression of proinflammatory cytokines and kynurenine pathway molecules was investigated by real-time RT-PCR on both unstimulated and stimulated PBMCs. The cows were retrospectively divided into healthy and diseased, based on the development of peripartum diseases (subclinical ketosis and placenta retention). The comparison between healthy and diseased cows suggested that healthy animals seemed to better control the response to LPS. On the contrary, diseased animals showed a much higher inflammatory response to LPS. Moreover, cows were retrospectively classified as high and low responders based on the in vitro proliferative response of PBMCs to LPS, using the median value as a threshold. Unstimulated PBMCs of low responders showed higher expression of the proinflammatory cytokines Interleukin 1-beta (IL-1 beta), Interleukin 6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-alpha), compared to high responders. Our preliminary data suggest that, during the peripartum period, high responders seem to be more tolerant to endotoxins and develop a lower inflammatory response to different stressors. Instead, low responders could be more prone to the development of unwanted inflammatory conditions in response to mild/moderate stressors

Phylogeny and Differential Gene Expression Profile of IL-1R8 in Normal Dog Tissue

Interleukin-1 Receptor 8 (IL-1R8) is a transmembrane protein of the IL-1 receptor family and represents an important regulator of the balance of innate and inflammatory responses. Depending on the immunological insult, IL1-R8 protects from the immunopathology or impairs the protective immune response against the insult. The expression pattern of IL-1R8 in dog tissues is unknown. Given the relevance of inflammatory diseases in dog, the aims of this study were to identify the sequence, analyze the phylogenesis and investigate the differential expression and distribution of IL-1R8 in a wide panel of non-pathologic tissues and organs by means of quantitative Real-Time PCR and uncover species-specific peculiarities. In Canis lupus familiaris, the IL1-R8 gene maps on chromosome 18, and includes ten exons. We first compared the coding sequence of dog IL-1R8 with sequences of other carnivors. Phylogenetic analysis revealed that IL-1R8 shares significantly high sequence homology with IL-1R8 of other canids particularly fox, sharing a common progenitor. Our study demonstrated that IL-1R8 is highly expressed in pancreas, considerably expressed in kidney, heart, liver, skeletal muscle, thymus, salivary gland, lymph node and lung. Interestingly, the expression pattern disclosed a unique profile for canine tissues when compared to tissues from other animal’s species. Imbalance of pro-inflammatory response leads to a vicious loop whither pro-inflammatory signaling and injury sustain each other and booster the disease. Therefore, it is crucial to investigate key regulator molecules such as IL-1R8, which functions both in homeostasis and disease and has potential to be a valid diagnostic, prognostic and therapeutic biomarker

IL-1R8 as Pathoimmunological Marker for Severity of Canine Chronic Enteropathy

Chronic enteropathy (CE) is a severe multifactorial gastrointestinal disease that affects dogs and is driven by poorly characterized inflammatory pathways. Imbalance of pro-inflammatory response regulators, including IL-1R8, may be due to different factors, among which the infection with Helicobacteraceae is known to lead to a vicious circle in which excessive pro-inflammatory signaling and gastrointestinal injury reinforce each other and boost the disease. We investigated the expression of IL-1R8 in large intestine biopsies of dogs with or without clinical signs of CE and with previously assessed enterohepatic Helicobacter spp. colonization status by mean of quantitative real-time PCR. Our study revealed that IL-1R8 is downregulated in both acutely (p = 0.0074) and chronically (p = 0.0159) CE affected dogs compared to healthy controls. The data also showed that IL-1R8 expression tends to decrease with colonization by Helicobacter spp. Interestingly, a negative correlation was detected between the level of expression of IL-1R8 and the severity of macroscopic lesions identified by endoscopy and the crypt hyperplasia score. We further compared the expression levels between males and females and found no statistically significant difference between the two groups. No significant difference was observed in IL-1R8 expression profiles with the age of the animals either. Interestingly, an association was uncovered between IL-1R8 expression level and dog breed. Together, our data advance knowledge on gastrointestinal pathoimmunology in dogs and highlight the potential utilization of IL-1R8 as a diagnostic, prognostic and therapeutic biomarker for canine chronic enteropathy

Preliminary Evidence of Endotoxin Tolerance in Dairy Cows during the Transition Period

The blastogenic response of bovine peripheral blood mononuclear cells (PBMCs) to lipopolysaccharides (LPS) has been investigated for a long time in our laboratories. In particular, a possible correlation between the blastogenic response to LPS and the disease resistance of dairy cows has been suggested in previous studies. Isolated PBMCs from eight cows at three different time points during the transition period (T0 = 15 days before calving; T1 = 7 days post-calving; T2 = 21 days post-calving) were cultured in the presence or absence of LPS, and the blastogenic response was assayed 72 h after in vitro stimulation. Moreover, the gene expression of proinflammatory cytokines and kynurenine pathway molecules was investigated by real-time RT-PCR on both unstimulated and stimulated PBMCs. The cows were retrospectively divided into healthy and diseased, based on the development of peripartum diseases (subclinical ketosis and placenta retention). The comparison between healthy and diseased cows suggested that healthy animals seemed to better control the response to LPS. On the contrary, diseased animals showed a much higher inflammatory response to LPS. Moreover, cows were retrospectively classified as high and low responders based on the in vitro proliferative response of PBMCs to LPS, using the median value as a threshold. Unstimulated PBMCs of low responders showed higher expression of the proinflammatory cytokines Interleukin 1-β (IL-1β), Interleukin 6 (IL-6) and Tumor Necrosis Factor-α (TNF-α), compared to high responders. Our preliminary data suggest that, during the peripartum period, high responders seem to be more tolerant to endotoxins and develop a lower inflammatory response to different stressors. Instead, low responders could be more prone to the development of unwanted inflammatory conditions in response to mild/moderate stressors

IL-1R8 Downregulation and Concomitant TLR7 and TLR9 Upregulation Are Related to the Pathogenesis of Canine Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common hematological malignancy in humans and dogs. Several studies disclosed some similarities between the two species, including the constitutive activation of the NF-κB pathway as a fundamental underlying pathogenetic mechanism. In humans, the downregulation of IL-1R8 is implicated in DLBCL development, but its role in dogs has not been explored so far. To gain insight into the pathogenesis of this tumor in dogs, we evaluated the mRNA and protein expression of IL-1R8 in 12 control lymph nodes obtained from dogs not bearing tumors and from 50 dogs with DLBCL. Moreover, we analyzed through qRT-PCR the expression of TLR7, TLR9, MYC, and p52 genes that are known to be involved in the IL-1R8 regulatory network. IL-1R8 and p52 were downregulated in DLBCLs compared to control lymph nodes (p < 0.001), while a higher expression of TLR7, TLR9, and MYC was observed in DLBCLs (p < 0.01). Immunohistochemistry confirmed the gene expression results, revealing a significantly lower IL-1R8 staining score in DLBCLs compared to control lymph nodes (p < 0.0001). Taken together, these results suggest that IL-1R8 downregulation may represent one of the mechanisms driving DLBCL pathogenesis in dogs, mainly through the dysregulation of the Toll-like/interleukin receptors signaling cascade and the aberrant activation of the classical NF-κB pathway

Bovine Colostrum Supplementation in Rabbit Diet Modulates Gene Expression of Cytokines, Gut–Vascular Barrier, and Red-Ox-Related Molecules in the Gut Wall

Rabbits, pivotal in the EU as livestock, pets, and experimental animals, face bacterial infection challenges, prompting a quest for alternatives to curb antibiotic resistance. Bovine colostrum (BC), rich in immunoregulatory compounds, antimicrobial peptides, and growth factors, is explored for disease treatment and prevention. This study assesses BC diet supplementation effects on rabbit intestines, examining gene expression. Thirty female New Zealand White rabbits at weaning (35 days) were divided into three experimental groups: control (commercial feed), 2.5% BC, and 5% BC. The diets were administered until slaughtering (81 days). BC-upregulated genes in the jejunum included IL-8, TGF-β, and CTNN-β1 at 5% BC, while PLVAP at 2.5% BC. Antioxidant-related genes (SOD1, GSR) were downregulated in the cecum and colon with 2.5% BC. BC 5% promoted IL-8 in the jejunum, fostering inflammation and immune cell migration. It also induced genes regulating inflammatory responses (TGF-β) and gastrointestinal permeability (CTNN-β1). BC 5% enhanced antioxidant activity in the cecum and colon, but no significant impact on anti-myxo antibody production was observed. These results suggest that BC has significant effects on the rabbit gastrointestinal tract’s inflammatory and antioxidant response, but further research is required to fully understand its histological and physiological impact

Epithelioid Pleural Mesothelioma Is Characterized by Tertiary Lymphoid Structures in Long Survivors: Results from the MATCH Study

Pleural mesothelioma (PM) is an aggressive tumor with few therapeutic options. Although patients with epithelioid PM (ePM) survive longer than non-epithelioid PM (non-ePM), heterogeneity of tumor response in ePM is observed. The role of the tumor immune microenvironment (TIME) in the development and progression of PM is currently considered a promising biomarker. A few studies have used high-throughput technologies correlated with TIME evaluation and morphologic and clinical data. This study aimed to identify different morphological, immunohistochemical, and transcriptional profiles that could potentially predict the outcome. A retrospective multicenter cohort of 129 chemonaive PM patients was recruited. Tissue slides were reviewed by dedicated pathologists for histotype classification and immunophenotype of tumor-infiltrating lymphocytes (TILs) and lymphoid aggregates or tertiary lymphoid structures (TLS). ePM (n = 99) survivors were further classified into long (>36 months) or short (<12 months) survivors. RNAseq was performed on a subset of 69 samples. Distinct transcriptional profiling in long and short ePM survivors was found. An inflammatory background with a higher number of B lymphocytes and a prevalence of TLS formations were detected in long compared to short ePM survivors. These results suggest that B cell infiltration could be important in modulating disease aggressiveness, opening a pathway for novel immunotherapeutic approaches