The identification of markers of macrophage differentiation in PMA-stimulated THP-1 Cells and monocyte-derived macrophages

Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD3) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD3 and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells

Monocytes regulate the mechanism of T-cell death by inducing Fas-mediated apoptosis during bacterial infection.

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed 'classic' features of apoptosis following exposure to pneumococci. Conversely, purified CD3(+) T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3(+) T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3(+) T-cells in PBMC cultures required 'classical' CD14(+) monocytes, which enhanced T-cell activation. CD3(+) T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3(+) T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease

Does autonomous macrophage-driven inflammation promote alveolar damage in COVID-19?

SARS-CoV-2 has caused devastating effects with over 550 million infections by July 2022 and approximately 6.4 million deaths [1]. Societal and economic impacts will reverberate for years, with continuous evolution of SARS-CoV-2 as it persistently spreads through the human population as exemplified by reduced activity of vaccines and monoclonals against Omicron BA.4 or BA.5 subvariants [2]. A greater understanding of pathogenesis and more tailored therapeutic approaches are therefore essential

Upper respiratory tract mucosal immunity for SARS-CoV-2 vaccines

SARS-CoV-2 vaccination significantly reduces morbidity and mortality, but has less impact on viral transmission rates, thus aiding viral evolution; and the longevity of vaccine-induced immunity rapidly declines. Immune responses in respiratory tract mucosal tissues are crucial for early control of infection, and can generate long-term antigen-specific protection with prompt recall responses. However, currently approved SARS-CoV-2 vaccines are not amenable to adequate respiratory mucosal delivery, particularly in the upper airways, which could account for the high vaccine breakthrough infection rates and limited duration of vaccine-mediated protection. In view of these drawbacks, we outline a strategy that has the potential to enhance both the efficacy and durability of existing SARS-CoV-2 vaccines, by inducing robust memory responses in the upper respiratory tract mucosa

Managing pneumonia through facility-based Integrated Management of Childhood Management (IMCI) Services:An analysis of the service availability and readiness among public health facilities in Bangladesh

Background With an estimated 24,000 deaths per year, pneumonia is the single largest cause of death among young children in Bangladesh, accounting for 18% of all under-5 deaths. The Government of Bangladesh adopted the WHO recommended Integrated Management of Childhood Illness (IMCI)-strategy in 1998 for outpatient management of pneumonia, which was scaled-up nationally by 2014. This paper reports the service availability and readiness related to IMCI-based pneumonia management in Bangladesh. We conducted a secondary analysis of the Bangladesh Health Facility Survey-2017, which was conducted with a nationally representative sample including all administrative divisions and types of health facilities. We limited our analysis to District Hospitals (DHs), Maternal and Child Welfare Centres (MCWCs), Upazila (sub-district) Health Complexes (UHCs), and Union Health and Family Welfare Centres (UH&FWCs), which are mandated to provide IMCI services. Readiness was reported based on 10 items identified by national experts as ‘essential’ for pneumonia management. Results More than 90% of DHs and UHCs, and three-fourths of UH&FWCs and MCWCs provide IMCI-based pneumonia management services. Less than two-third of the staff had ever received IMCI-based pneumonia training. Only one-third of the facilities had a functional ARI timer or a watch able to record seconds on the day of the visit. Pulse oximetry was available in 27% of the district hospitals, 18% of the UHCs and none of the UH&FWCs. Although more than 80% of the facilities had amoxicillin syrup or dispersible tablets, only 16% had injectable gentamicin. IMCI service registers were not available in nearly one-third of the facilities and monthly reporting forms were not available in around 10% of the facilities. Only 18% of facilities had a high-readiness (score 8–10), whereas 20% had a low-readiness (score 0–4). The readiness was significantly poorer among rural and lower level facilities (p < 0.001). Seventy-two percent of the UHCs had availability of one of any of the four oxygen sources (oxygen concentrators, filled oxygen cylinder with flowmeter, filled oxygen cylinder without flowmeter, and oxygen distribution system) followed by DHs (66%) and MCWCs (59%). Conclusion There are substantial gaps in the readiness related to IMCI-based pneumonia management in public health facilities in Bangladesh. Since pneumonia remains a major cause of child death nationally, Bangladesh should make a substantial effort in programme planning, implementation and monitoring to address these critical gaps to ensure better provision of essential care for children suffering from pneumonia

Roles of neutrophils in the regulation of the extent of human inflammation through delivery of IL-1 and clearance of chemokines

This study examined the establishment of neutrophilic inflammation in humans. We tested the hypotheses that neutrophil recruitment was associated with local CXCL8 production and that neutrophils themselves might contribute to the regulation of the size of the inflammatory response. Humans were challenged i.d. with endotoxin. Biopsies of these sites were examined for cytokine production and leukocyte recruitment by qPCR and IHC. Additional in vitro models of inflammation examined the ability of neutrophils to produce and sequester cytokines relevant to neutrophilic inflammation. i.d. challenge with 15 ng of a TLR4-selective endotoxin caused a local inflammatory response, in which 1% of the total biopsy area stained positive for neutrophils at 6 h, correlating with 100-fold up-regulation in local CXCL8 mRNA generation. Neutrophils themselves were the major source of the early cytokine IL-1β. In vitro, neutrophils mediated CXCL8 but not IL-1β clearance (>90% clearance of ≤2 nM CXCL8 over 24 h). CXCL8 clearance was at least partially receptor-dependent and modified by inflammatory context, preserved in models of viral infection but reduced in models of bacterial infection. In conclusion, in a human inflammatory model, neutrophils are rapidly recruited and may regulate the size and outcome of the inflammatory response through the uptake and release of cytokines and chemokines in patterns dependent on the underlying inflammatory stimulus

Hypoxaemia prevalence and its adverse clinical outcomes among children hospitalised with WHO-defined severe pneumonia in Bangladesh

BACKGROUND: With an estimated 1 million cases per year, pneumonia accounts for 15% of all under-five deaths globally, and hypoxaemia is one of the strongest predictors of mortality. Most of these deaths are preventable and occur in low- and middle-income countries. Bangladesh is among the six high burden countries with an estimated 4 million pneumonia episodes annually. There is a gap in updated evidence on the prevalence of hypoxaemia among children with severe pneumonia in high burden countries, including Bangladesh. METHODS: We conducted a secondary analysis of data obtained from icddr,b-Dhaka Hospital, a secondary level referral hospital located in Dhaka, Bangladesh. We included 2646 children aged 2-59 months admitted with WHO-defined severe pneumonia during 2014-17. The primary outcome of interest was hypoxaemia, defined as SpO(2) < 90% on admission. The secondary outcome of interest was adverse clinical outcomes defined as deaths during hospital stay or referral to higher-level facilities due to clinical deterioration. RESULTS: On admission, the prevalence of hypoxaemia among children hospitalised with severe pneumonia was 40%. The odds of hypoxaemia were higher among females (adjusted Odds ratio AOR = 1.44; 95% confidence interval CI = 1.22-1.71) and those with a history of cough or difficulty in breathing for 0-48 hours before admission (AOR = 1.61; 95% CI = 1.28-2.02). Among all children with severe pneumonia, 6% died during the hospital stay, and 9% were referred to higher-level facilities due to clinical deterioration. Hypoxaemia was the strongest predictor of mortality (AOR = 11.08; 95% CI = 7.28-16.87) and referral (AOR = 5.94; 95% CI = 4.31-17) among other factors such as age, sex, history of fever and cough or difficulty in breathing, and severe acute malnutrition. Among those who survived, the median duration of hospital stay was 7 (IQR = 4-11) days in the hypoxaemic group and 6 (IQR = 4-9) days in the non-hypoxaemic group, and the difference was significant at P < 0.001. CONCLUSIONS: The high burden of hypoxaemia and its clinical outcomes call for urgent attention to promote oxygen security in low resource settings like Bangladesh. The availability of pulse oximetry for rapid identification and an effective oxygen delivery system for immediate correction should be ensured for averting many preventable deaths

Macrophage phenotype is associated with disease severity in preterm infants with chronic lung disease.

The etiology of persistent lung inflammation in preterm infants with chronic lung disease of prematurity (CLD) is poorly characterized, hampering efforts to stratify prognosis and treatment. Airway macrophages are important innate immune cells with roles in both the induction and resolution of tissue inflammation