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

Common, low-frequency, rare, and ultra-rare coding variants contribute to COVID-19 severity

The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole-exome sequencing data of about 4000 SARS-CoV-2-positive individuals were used to define an interpretable machine-learning model for predicting COVID-19 severity. First, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthily, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management. © 2021, The Author(s)

Local atmospheric circulations and the mesoclimate of Durban.

Thesis (Ph.D.)-University of Natal, 1970.Rapid urban and industrial growth along the Natal coast has occurred with little concern for characteristics of local climate. In general, industrial growth has taken place without taking into account the potential for pollution transportation during the winter season; urban areas have developed without consideration for the vital need in sub-tropical latitudes to maintain adequate mixing of the lower atmosphere. The apparent inadequacy in planning for climate has partly been due to a lack of understanding of the nature and characteristics of local wind systems. The motivation for this study stems , therefore, from the need to evaluate the influence of land-sea and topographically-induced wind systems upon certain aspects of weather and climate on the Natal coast. During summer, moist and relatively cool air is advected almost daily over the Natal coast by the sea breeze . Observations of spatial and temporal variations of these winds were made in the period 1963-69 from recording stations in the Durban area and along a 40-mile transect approximately normal to the coast at Durban. Comparable observations have not as yet been made in South Africa nor have there been a ttempts to examine the influence of sea breezes upon selected climate and weather phenomenon. In this latter connection the role of the sea br eeze is examined in two separate fields in this study, namely urban climatology and thunderstorm development. Climatic discomfort caused by the combined effect of high temperatures and humidities on the Natal coast, is at a maximum in urban areas . Since these areas are also highly populated , a need exists to evaluate the ability of the sea breeze to reduce urban temperatures by advection of cooler air over the area, by eddy diffusion of heat or by displacement of urban temperatures aw~ from poorly ventilated areas. Thus climatic discomfort in these areas may also be reduced. Detailed observations of the influence of the sea breeze, in particular, and wind in general, upon the spatial variation of temperature , humidity and human comfort were, therefore, undertaken in the Durban area. It is believed that this i s the first time an attempt has been made to describe the spatial distribution of a comfort index in an urban area. The results should be of interest to the a rchitect and town planner. The sea breeze, strengthened by gradient and valley Vlinds, is shown to penetrate at least 40 miles inland. In accordance with this , it is suggested that the inland invasion of marine air takes place in response to lowered pressure in adjacent inland areas in southern Natal and provides the moisture required to feed thunderstorms which develop in this area. These storms subsequently move towards the coast following the retreating convergence zone between winds with an onshore and offshore component of motion and reach Durban after sunset. Land breezes are best developed in winter. Observational techniques were similar to those employed in the sea breeze study and provide the only detailed examination of spatial and temporal variations of land breezes in South Africa. Until recently the nature and characteristics of topographically-induced winds in Natal were also a relatively unexplored feature of local atmospheric circulations. However, Tyson (1967) has examined local winds in certain Natal valleys and his prediction that mountain- plain winds reach the coast during the night is also examined in this study. While the relatively weak. and shallow land breeze may be of secondary importance when compared with the sea breeze, it would be a mistake to underestimate the importance of these winds upon certain aspects of coastal climate. In p~rticular the tendency for land breezes to initiate cloud and precipitation at night is examined while the potential for the transportation of atmospheric pollution by these winds is also briefly discussed. A study of the diurnal variation of precipitation at Durban reveals a high frequency of low intensi~ precipitation at night. The nature of this precipitation differs in both frequency and" amount from high intensity rainfall which is the product of thunderstorm and frontal activity. It is suggested in this study that the land breeze plays a dominant role in providing the buoyancy necessary to cause nocturnal drizzle precipitation from shallow stratus cloud. The thesis is divided into four parts. Part I examines details of the physiography, weather, climate, observati ~nal methods and analysis techniques. Because of the variability of weather on the coast of Natal that section is dealt with in more detail than would have been deemed necessary for a similar study inland. Without knowledge of the characteristics of the atmospheric circulation, the behaviour of local wind systems in relation to large scale systems could not be adequately discussed. In Part II land and sea breezes and topographically-induced wind systems are examined in detail. Emphasis is placed not only on the observational characteristics of these winds such as onset, depth, velocity gradient, surging, relation to gradient winds and dissipation but also on their relation to theoretical models of the relevant wind system. The influence of local wind systems upon selected climatic elements is dealt with in Part Ill. Characteristics of the diurnal variation of precipitation are examined in Chapter 6. Rainfall frequencies and amounts are shown to be highest in the first half of the night and a model is developed to explain the influence of land breezes and mountain-plain winds upon low intensity but high frequency nocturnal rainfall. High intensity but low frequency rainfall produced by thunderstorms also occurs soon after sunset and a further model is advanced to explain this phenomenon in relation to the sea breeze. The effect of air movement upon the spatial variation of temperature, humidity and a discomfort index in both summer and winter is examined in Chapters 7 and 8. The relationship between these elements has permitted the development of an empirical model to predict values across the city of mean midday summer and winter temperatures as well as discomfort index values. In Part IV the most significant aspeots relating to the study as a whole are summarised. In conclusion the potential f or the transport of pollutants by land breezes and topographically induced winds is briefly discussed as an aid to planning and siaple spatial models are given showing generalised air movement and ventilation characteristics in the Durban area and along the Natal coast

Pneumolysin activates macrophage lysosomal membrane permeabilization and executes apoptosis by distinct mechanisms without membrane pore formation

Intracellular killing of Streptococcus pneumoniae is complemented by induction of macrophage apoptosis. Here, we show that the toxin pneumolysin (PLY) contributes both to lysosomal/phagolysosomal membrane permeabilization (LMP), an upstream event programing susceptibility to apoptosis, and to apoptosis execution via a mitochondrial pathway, through distinct mechanisms. PLY is necessary but not sufficient for the maximal induction of LMP and apoptosis. PLY's ability to induce both LMP and apoptosis is independent of its ability to form cytolytic pores and requires only the first three domains of PLY. LMP involves TLR (Toll-like receptor) but not NLRP3/ASC (nucleotide-binding oligomerization domain [Nod]-like receptor family, pyrin domain-containing protein 3/apoptosis-associated speck-like protein containing a caspase recruitment domain) signaling and is part of a PLY-dependent but phagocytosis-independent host response that includes the production of cytokines, including interleukin-1 beta (IL-1β). LMP involves progressive and selective permeability to 40-kDa but not to 250-kDa fluorescein isothiocyanate (FITC)-labeled dextran, as PLY accumulates in the cytoplasm. In contrast, the PLY-dependent execution of apoptosis requires phagocytosis and is part of a host response to intracellular bacteria that also includes NO generation. In cells challenged with PLY-deficient bacteria, reconstitution of LMP using the lysomotrophic detergent LeuLeuOMe favored cell necrosis whereas PLY reconstituted apoptosis. The results suggest that PLY contributes to macrophage activation and cytokine production but also engages LMP. Following bacterial phagocytosis, PLY triggers apoptosis and prevents macrophage necrosis as a component of a broad-based antimicrobial strategy. This illustrates how a key virulence factor can become the focus of a multilayered and coordinated innate response by macrophages, optimizing pathogen clearance and limiting inflammation. Importance: Streptococcus pneumoniae, the commonest cause of bacterial pneumonia, expresses the toxin pneumolysin, which can make holes in cell surfaces, causing tissue damage. Macrophages, resident immune cells essential for responses to bacteria in tissues, activate a program of cell suicide called apoptosis, maximizing bacterial clearance and limiting harmful inflammation. We examined pneumolysin's role in activating this response. We demonstrate that pneumolysin did not directly form holes in cells to trigger apoptosis and show that pneumolysin has two distinct roles which require only part of the molecule. Pneumolysin and other bacterial factors released by bacteria that have not been eaten by macrophages activate macrophages to release inflammatory factors but also make the cell compartment containing ingested bacteria leaky. Once inside the cell, pneumolysin ensures that the bacteria activate macrophage apoptosis, rather than necrosis, enhancing bacterial killing and limiting inflammation. This dual response to pneumolysin is critical for an effective immune response to S. pneumoniae.</p

Distinct cell death programs in monocytes regulate innate responses following challenge with common causes of invasive bacterial disease

Peripheral blood monocytes represent the rapid response component of mononuclear phagocyte host defense, generating vigorous but finite antibacterial responses. We investigated the fate of highly purified primary human monocytes following phagocytosis of different bacteria. Exposure to high bacterial loads resulted in rapid loss of cell viability and decreased functional competence. Cell death typically involved classical apoptosis. Exposure to high numbers of Escherichia coli and Klebsiella pneumoniae induced nonapoptotic death with loss of cell membrane integrity, marked disruption of phagolysosomes, and caspase-1 activation, while a subset of cells also released caspase-1-regulated extracellular traps. Classical apoptosis increased if extracellular bacterial replication was reduced and decreased if intracellular ATP levels were reduced during these infections. Both classical apoptosis and the alternative forms of cell death allowed monocytes, whose functional competence was exhausted, to downregulate reactive oxygen species and proinflammatory cytokine responses. In contrast, sustained stimulation of glycolytic metabolism and mitochondrial oxidative phosphorylation, with associated hypoxia inducible factor-1alpha upregulation, maintained intracellular ATP levels and prolonged monocyte functional longevity, as assessed by maintenance of phagocytosis, reactive oxygen species production, and proinflammatory cytokine generation. Monocyte innate responses to bacteria are short-lived and are limited by an intrinsic program of apoptosis, a response that is subverted by overwhelming infection with E. coli and K. pneumoniae or bacterial stimulation of cell metabolism. In this regard, the fate of monocytes following bacterial challenge more closely resembles neutrophils than macrophages

Purified T-cells undergo a necrotic pneumolysin-dependent death.

<p>A) Representative transmission electron micrograph of CD3⁺ T-cells from peripheral blood mononuclear cells (PBMC) and CD3 enriched cultures (CD3 enriched) following challenge with D39 <i>Streptococcus pneumoniae</i> (MOI = 50) or mock-infection (MI) for 6 h. CD3⁺ T-cells from PBMC cultures showed characteristics of apoptosis including nuclear fragmentation while CD3 enriched T-cells had features of necrosis such as membrane rupture. Images were obtained with an FEI Tecnai transmission electron microscope. B) T-cell trypan blue exclusion was assessed by brightfield microscopy following challenge of PBMC and CD3 enriched cultures with D39 (MOI = 50) for 6 h and C) pre-treatment with necrostatin-1 (30 nM) under the same conditions as B) did not reduce CD3⁺ death measured by TO-PRO-3 staining or D) convert the death process in CD3 enriched cultures to apoptosis, measured by accumulation of hypodiploid DNA (% Sub G0/1), n = 4. E) The mean percentage of Annexin V⁺ CD3⁺ T-cells 6 h after mock-infection (MOI = 0) or challenge of PBMC with D39 or the Δ6 mutant (MOI = 50), n = 5. F) The mean percentage of Annexin V⁺ and G) % sub G0/1peripheral blood lymphocytes isolated by plastic adherence of PBMC and gated by FSC and SSC 6 h following challenge with pneumolysin (0–1 µg), n = 3 *p<0.05, **p<0.01, ***p<0.001; statistical analysis by one and two-way ANOVA or t-test.</p

CD3⁺ T-cells undergo Fas-mediated apoptosis following pneumococcal challenge.

<p>A) Mean caspase 8 relative luminescence units (Caspase 8 RLU) in CD3⁺ T-cells from peripheral blood mononuclear cells (PBMC), purified CD3⁺ T-cells (CD3+ CD14−) or co-cultures of purified CD3⁺ T-cells with 10% purified CD14⁺ monocytes (CD3+ CD14+) 6 h following mock-infection (multiplicity of infection (MOI) = 0) or challenge with D39 <i>Streptococcus pneumoniae</i> (MOI = 50), n = 5. B) Representative western blot probed for Bid, Bim and actin from CD3⁺ T-cells purified from PBMC 16 h following mock-infection (D39−) or challenge with D39 at MOI = 0.1 (D39+) in the presence of isotype control (Isotype+) or ZB4 neutralizing anti-Fas antibody (Anti-Fas+) added at 1 µg/ml. C) Densitometry summarises the fold change in Bid compared to the mock infected (MI) cells from three separate experiments with different donors. D) Cytosolic and membrane fractions were also probed for cytochrome c, actin and Cox-4, and E) densitometry was performed, n = 4. F) Hypodiploid DNA accumulation (Sub G0/1) was measured in CD3⁺ T-cells in PBMC cultured under the same conditions as B), n = 4. G) The percentage of Annexin V⁺ CD3⁺ T-cells in PBMC cultured under the same condition as in B), n = 3, * p<0.05; statistical analysis by ANOVA or t-test.</p