Single-Cell Imaging Maps Inflammatory Cell Subsets to Pulmonary Arterial Hypertension Vasculopathy.

Rationale: Unraveling immune-driven vascular pathology in pulmonary arterial hypertension (PAH) requires a comprehensive understanding of the immune cell landscape. Although patients with hereditary (H)PAH and bone morphogenetic protein receptor type 2 (BMPR2) mutations have more severe pulmonary vascular pathology, it is not known whether this is related to specific immune cell subsets. Objectives: This study aims to elucidate immune-driven vascular pathology by identifying immune cell subtypes linked to severity of pulmonary arterial lesions in PAH. Methods: We used cutting-edge multiplexed ion beam imaging by time of flight to compare pulmonary arteries (PAs) and adjacent tissue in PAH lungs (idiopathic [I]PAH and HPAH) with unused donor lungs, as controls. Measurements and Main Results: We quantified immune cells' proximity and abundance, focusing on those features linked to vascular pathology, and evaluated their impact on pulmonary arterial smooth muscle cells (SMCs) and endothelial cells. Distinct immune infiltration patterns emerged between PAH subtypes, with intramural involvement independently linked to PA occlusive changes. Notably, we identified monocyte-derived dendritic cells within PA subendothelial and adventitial regions, influencing vascular remodeling by promoting SMC proliferation and suppressing endothelial gene expression across PAH subtypes. In patients with HPAH, pronounced immune dysregulation encircled PA walls, characterized by heightened perivascular inflammation involving T cell immunoglobulin and mucin domain-3 (TIM-3)+ T cells. This correlated with an expanded DC subset expressing indoleamine 2,3-dioxygenase 1, TIM-3, and SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1, alongside increased neutrophils, SMCs, and alpha-smooth muscle actin (ACTA2)+ endothelial cells, reinforcing the heightened severity of pulmonary vascular lesions. Conclusions: This study presents the first architectural map of PAH lungs, connecting immune subsets not only with specific PA lesions but also with heightened severity in HPAH compared with IPAH. Our findings emphasize the therapeutic potential of targeting monocyte-derived dendritic cells, neutrophils, cellular interactions, and immune responses to alleviate severe vascular pathology in IPAH and HPAH.

Multiplexed imaging reveals an IFN-γ-driven inflammatory state in nivolumab-associated gastritis.

Immune checkpoint blockade using PD-1 inhibition is an effective approach for treating a wide variety of cancer subtypes. While lower gastrointestinal (GI) side effects are more common, upper gastrointestinal adverse events are rarely reported. Here, we present a case of nivolumab-associated autoimmune gastritis. To elucidate the immunology underlying this condition, we leverage multiplexed ion beam imaging by time-of-flight (MIBI-TOF) to identify the presence and proportion of infiltrating immune cells from a single section of biopsy specimen. Using MIBI-TOF, we analyze formalin-fixed, paraffin-embedded human gastric tissue with 28 labels simultaneously. Our analyses reveal a gastritis characterized by severe mucosal injury, interferon gamma (IFN-γ)-producing gastric epithelial cells, and mixed inflammation that includes CD8 and CD4 T cell infiltrates with reduced expression of granzyme B and FOXP3, respectively. Here, we provide a comprehensive multiplexed histopathological mapping of gastric tissue, which identifies IFN-γ-producing epithelial cells as possible contributors to the nivolumab-associated gastritis.

Impact of chemokine C-C ligand 27, foreskin anatomy and sexually transmitted infections on HIV-1 target cell availability in adolescent South African males.

We compared outer and inner foreskin tissue from adolescent males undergoing medical male circumcision to better understand signals that increase HIV target cell availability in the foreskin. We measured chemokine gene expression and the impact of sexually transmitted infections (STIs) on the density and location of T and Langerhans cells. Chemokine C-C ligand 27 (CCL27) was expressed 6.94-fold higher in the inner foreskin when compared with the outer foreskin. We show that the density of CD4+CCR5+ cells/mm2 was higher in the epithelium of the inner foreskin, regardless of STI status, in parallel with higher CCL27 gene expression. In the presence of STIs, there were higher numbers of CD4+CCR5+ cells/mm2 cells in the sub-stratum of the outer and inner foreskin with concurrently higher number of CD207+ Langerhans cells (LC) in both tissues, with the latter cells being closer to the keratin surface of the outer FS in the presence of an STI. When we tested the ability of exogenous CCL27 to induce T-cell migration in foreskin tissue, CD4 + T cells were able to relocate to the inner foreskin epithelium in response. We provide novel insight into the impact CCL27 and STIs on immune and HIV-1 target cell changes in the foreskin.

Frequency of Circulating CD4+Ki67+HLA-DR- T Regulatory Cells Prior to Treatment for Multidrug Resistant Tuberculosis Can Differentiate the Severity of Disease and Predict Time to Culture Conversion.

Identifying a blood circulating cellular biomarker that can be used to assess severity of disease and predict the time to culture conversion (TCC) in patients with multidrug resistant tuberculosis (MDR-TB) would facilitate monitoring response to treatment and may be of value in the design of future drug trials. We report on the frequency of blood Ki67+HLA-DR- CD4+ T regulatory (Treg) cells in predicting microbiological outcome before initiating second-line treatment for MDR-TB. Fifty-one patients with MDR-TB were enrolled and followed over 18 months; a subset of patients was sputum culture (SC) negative at baseline (n = 9). SC positive patients were divided into two groups, based on median TCC: rapid responders (≤71 days TCC; n = 21) and slow responders (>71 days TCC; n = 21). Whole blood at baseline, months 2 and 6 was stimulated with M tuberculosis (Mtb) antigens and Treg cells were then identified as CD3+CD4+CD25hiFoxP3+CD127-CD69- and further delineated as Ki67+HLA-DR- Treg. The frequency of these cells was significantly enlarged at baseline in SC positive relative to SC negative and smear positive relative to smear negative patients and in those with lung cavitation. This difference was further supported by unsupervised hierarchical clustering showing a significant grouping at baseline of total and early differentiated memory Treg cells in slow responders. Conversely, there was a clustering of a lower proportion of Treg cells and activated IFNγ-expressing T cells at baseline in the rapid responders. Examining changes over time revealed a more gradual reduction of Treg cells in slow responders relative to rapid responders to treatment. Receiver operating curve analysis showed that baseline Mtb-stimulated Ki67+HLA-DR- Treg cells could predict the TCC of MDR-TB treatment response with 81.2% sensitivity and 85% specificity (AUC of 0.87, p < 0.0001), but this was not the case after 2 months of treatment. In conclusion, our data show that the frequency of a highly defined Mtb-stimulated blood Treg cell population at baseline can discriminate MDR-TB disease severity and predict time to culture clearance.

A combination of baseline plasma immune markers can predict therapeutic response in multidrug resistant tuberculosis.

OBJECTIVE: To identify plasma markers predictive of therapeutic response in patients with multidrug resistant tuberculosis (MDR-TB). METHODS: Fifty HIV-negative patients with active pulmonary MDR-TB were analysed for six soluble analytes in plasma at the time of initiating treatment (baseline) and over six months thereafter. Patients were identified as sputum culture positive or negative at baseline. Culture positive patients were further stratified by the median time to sputum culture conversion (SCC) as fast responders (< 76 days) or slow responders (≥ 76 days). Chest X-ray scores, body mass index, and sputum smear microscopy results were obtained at baseline. RESULTS: Unsupervised hierarchical clustering revealed that baseline plasma levels of IP-10/CXCL10, VEGF-A, SAA and CRP could distinguish sputum culture and cavitation status of patients. Among patients who were culture positive at baseline, there were significant positive correlations between plasma levels of CRP, SAA, VEGF-A, sIL-2Rα/CD40, and IP-10 and delayed SCC. Using linear discriminant analysis (LDA) and Receiver Operating Curves (ROC), we showed that a combination of MCP-1/CCL2, IP-10, sIL-2Rα, SAA, CRP and AFB smear could distinguish fast from slow responders and were predictive of delayed SCC with high sensitivity and specificity. CONCLUSION: Plasma levels of specific chemokines and inflammatory markers measured before MDR-TB treatment are candidate predictive markers of delayed SCC. These findings require validation in a larger study.

Host responses associated with chronic staphylococcal mastitis in rabbits.

Staphylococcal infection causes substantial economic losses in commercial rabbit production systems, and is associated with a wide variety of lesions, including chronic suppurative mastitis, which mainly affects breeding females. Most chronic staphylococcal infections in rabbits are caused by the ST121 lineage of Staphylococcus aureus, although other less common lineages, such as ST96 can also be involved. The aims of the present study were to characterise the host immune response in natural cases of mastitis in rabbits caused by S. aureus, to evaluate any relationship between peripheral and local immunity and to investigate the effect of different S. aureus genotypes on these immune responses. Adult multiparous female rabbits that were affected with chronic staphylococcal mastitis (n = 204) were enrolled into the study. Histological and immunohistochemical evaluations of mammary glands were undertaken, as well as flow cytometric analyses of blood. S. aureus isolates from the mammary glands were identified by multilocus sequence typing. Differences in the number of infiltrating cells were detected, depending on the type of pathology, with more immature lesions demonstrating greater cellularity, characterised by greater numbers of T lymphocytes, macrophages and plasma cells. A relationship was seen between the cells in blood and mammary tissues, the most notable being the positive correlation between monocytes and tissue macrophages. When glands were infected with ST96 strains, fewer granulocytes (P < 0.01) and greater numbers of B cells (P < 0.01), T cells (P < 0.001), CD4(+) T cells (P < 0.001) and CD8(+) T cells (P < 0.01) were detected, compared with mammary glands that were infected by ST121 strains of S. aureus.

How selection for reproduction or foundation for longevity could have affected blood lymphocyte populations of rabbit does under conventional and heat stress conditions.

The present work characterises how selection for reproduction (by comparing two generations - 16th and 36th - of the V line selected for litter size at weaning) or foundation for reproductive longevity (the LP line) can affect the blood lymphocytes populations of reproductive rabbit does under normal [conventional housing, average daily minimum and maximum temperatures of 14°C and 20°C, respectively] and heat stress conditions [climatic chamber, 25°C and 36°C] from the first to the second parturition. Housing under heat stress conditions significantly reduced the B lymphocytes counts in female rabbits (-34 × 10(6)/L; P<0.05). The highest lymphocytes population value in blood (total, T CD5(+), CD4(+) and CD8(+)) was noted at the first parturition, while the B lymphocytes count was significantly lower at the second parturition (-61 × 10(6)/L; P<0.05). Selection for litter size at weaning (V females) reduced the average counts of total and B lymphocytes in blood (-502 and -60 × 10(6)/L, respectively; P<0.01), mainly because these populations in V36 females continuously lowered from the first to the second parturition under normal housing conditions. Thus, more selected females (V36) at the second parturition showed significantly lower counts in blood for total, T CD5(+) and CD25(+) lymphocytes (-1303, -446 and -33 × 10(6)/L, respectively; P<0.05). The main differences in blood counts between V36 and V16 females disappeared when housed under heat stress conditions, except for T CD5(+) and CD25(+), which significantly increased (T CD5(+): +428 × 10(6)/L; CD25(+): +41 × 10(6)/L; P<0.01) in the V16 vs. V36 females on day 10 post-partum. Under normal conditions, no differences between LP and V36 females were found for most lymphocyte populations; only higher counts were noted in CD25(+) (+20 × 10(6)/L; P<0.05) for LP females. However, the lymphocytes counts [especially total (+1327 × 10(6)/L; P<0.01) and T CD5(+) (+376 × 10(6)/L; P<0.10)] of LP females increased under heat vs. normal conditions when lymphocytes populations presented the lowest values (second parturition), while V36 females' counts remained invariable. Positive correlations were found between feed intake (r=+0.51; P<0.001) and females' perirenal fat thickness (r=+0.40; P<0.001) with B lymphocytes counts in the blood of primiparous rabbit females in the week 2 of lactation. These results indicate that selection for litter size at weaning might diminish their immune system's response and adaptation capacity, while the foundation for reproductive longevity criteria leads to more robust rabbit females as they present greater modulation under heat stress conditions when the immune system is affected.

Evolution of the peripheral blood lymphocyte populations in multiparous rabbit does with two reproductive management rhythms.

The emergence of epizootic rabbit enteropathy is leading to changes in weaning protocols in commercial rabbitries. Traditional weaning protocols are being replaced with late weaning, beyond 35 days postpartum (dpp). The main objectives of this study were to compare the peripheral blood lymphocyte populations of multiparous rabbit does under two reproductive rhythms (insemination at 11 dpp and weaning at 28 dpp, insemination at 25 dpp and weaning at 42 dpp), and to assess the influence on those of kits. Samples of peripheral blood were taken in 22 adult females and 44 of their kits at different critical times, and several lymphocytic populations were evaluated by flow cytometry. Additionally, the perirenal fat thickness of does was also measured at partum and weaning to observe if body condition correlates with lymphocyte populations. During whole lactation, counts of total, CD5(+), CD4(+) and CD8(+) lymphocytes of females were generally lower with weaning at 42 dpp compared to 28 dpp. Moreover, counts of total, B and CD5(+) lymphocytes in rabbit does weaned at 42 dpp correlated to their body condition (+0.60 to 0.82; P<0.05), contrary to that observed in rabbit does weaned at 28 dpp. Some correlations between lymphocyte counts in both groups of does and weaning rabbits were observed. At weaning, those young rabbits weaned at 42 dpp had a significantly lower number of CD4(+) lymphocytes than those weaned at 28 dpp (P<0.01). In conclusion, the 42 ddp rabbit does presented a lower number of total lymphocytes and lymphocytic subpopulations during lactation and at weaning, as well as lesser capacity of adjustment during the gestation-lactation cycle.