Establishment of heterochromatin in domain-size-dependent bursts

Methylation of histone H3K9 is a hallmark of epigenetic silencing in eukaryotes. Nucleosome modifications often rely on positive feedback where enzymes are recruited by modified nucleosomes. A combination of local and global feedbacks has been proposed to account for some dynamic properties of heterochromatin, but the range at which the global feedbacks operate and the exact mode of heterochromatin propagation are not known. We investigated these questions in fission yeast. Guided by mathematical modeling, we incrementally increased the size of the mating-type region and profiled heterochromatin establishment over time. We observed exponential decays in the proportion of cells with active reporters, with rates that decreased with domain size. Establishment periods varied from a few generations in wild type to >200 generations in the longest region examined, and highly correlated silencing of two reporters located outside the nucleation center was observed. On a chromatin level, this indicates that individual regions are silenced in sudden bursts. Mathematical modeling accounts for these bursts if heterochromatic nucleosomes facilitate a deacetylation or methylation reaction at long range, in a distance-independent manner. A likely effector of three-dimensional interactions is the evolutionarily conserved Swi6^HP1 H3K9me reader, indicating the bursting behavior might be a general mode of heterochromatin propagation.

Histone-modifying enzymes covalently attach small polypeptides or chemical groups to nucleosomes, thereby controlling gene expression. Some modifications propagate from nucleation sites over large chromosomal distances. They can be maintained clonally upon cell division or overridden to allow for cell differentiation or adaptation to changing environments. Among the many histone modifications that define chromatin structure, the methylation of histone H3K9 maintains large chromosomal regions in a silent heterochromatic state.Modeling has pointed to key parameters in chromatin formation and maintenance, for instance, the importance of cooperativity in the recruitment of modifying enzymes to chromatin or how various types of positive feedback would contribute differentially to the properties of chromatin (1–4). Nucleosome interconversion models developed with one organism, fission yeast, have been applied to distant species such as Arabidopsis, Drosophila, or mice, transposed to other modifications, for example, from H3K9 to H3K27 methylation (2, 5–7), and merged with other forms of modeling (8). In Arabidopsis, such models have uncovered fundamental aspects of vernalization by showing how epigenetic memory is digitally acquired and stored in cis at the FLC locus for long-term gene silencing and by anticipating the sequence of events taking place at that locus (2, 4, 6). In Drosophila, nucleosome-based models shed new light on the concerted action of PRC complexes (7). Recently, molecular models have been also complemented by an operational model of chromatin-mediated silencing, which supports the view that silencing is a stochastic event that occurs in an all-or-none fashion in individual cells (9).A central element of nucleosome interconversion models is that they accommodate feedbacks arising through the direct or indirect recruitment of enzymes by modified nucleosomes. In nature, self-recruitment applies to many histone-modifying enzymes and enzymatic complexes (10–12), some of which might be able to modify nucleosomes brought into proximity by chromatin folding (13–15), while others would uniquely modify immediate neighbors. Hence, among the fundamental questions that quantitative modeling can address are the impacts of short- and long-range nucleosomal interactions occurring in the nucleus on chromatin dynamics. This is deeply connected to the overall expectation that heterochromatin stability depends on the size of the region in question.To explore these questions, we used here the mating-type region of the fission yeast Schizosaccharomyces pombe. In this system, heterochromatin formation entails methylation of histone H3K9 by a single Su(var)39-family enzyme, Clr4, in combination with nucleosomal deacetylation by various histone deacetylases (HDACs), but no DNA methylation. The region contains the silent mat2-P and mat3-M loci used in mating-type switching (16) and other elements (Fig. 1), including a central cenH element with homology to centromeric and subtelomeric repeats (17, 18).Open in a separate windowFig. 1.Domain-size-dependent establishment of heterochromatin. (A) Mating-type region of S. pombe showing two reporters used to monitor the establishment of heterochromatic silencing, YFP at the Kint2 site within the nucleation center cenH and mCherry at the (EcoRV) site. The heterochromatic domain is delimited by the IR-L and IR-R boundary elements and contains two Atf1 binding sites, s1 and s2. The region was extended by inserting, at the indicated Kint3 site, DNA originating from the leu1⁺ chromosomal region, respectively, a 4.5-kb, two distinct 5.9-kb, and a 7.8-kb fragment (see also SI Appendix, Fig. S1) generating mating-type regions of, respectively, 27.5 kb, 29 kb (SH4), and 31 kb. (B and C) The clr4⁺ gene was reintroduced into clr4Δ cells with extended mating-type regions (B) or original length (C) through genetic crosses, and the establishment of (EcoRV)::mCherry silencing was monitored in cultures maintained in exponential growth for 19 d, taking one time point per day. Overlays of brightfield and red fluorescence micrographs are presented for days 2, 4, 8, 12, and 17 as indicated. Histograms of cell fluorescence intensities are shown for the same cultures, each compiling >15,000 cells. Individual channels including YFP are shown in SI Appendix, Figs. S2–S4. (D) Two examples of clonal populations derived from the strain with the 31-kb mating-type region at day 17 and propagated for approximately 40 generations. Each micrograph shows a field 86.5 × 66 μm.Time course experiments have found that the nucleation of heterochromatin occurs at cenH, stochastically and at a high rate (3). It is driven by RNA interference (RNAi) (19, 20) as at centromeres (21–23). Outward expansion relies on positive feedback by several chromodomain proteins that bind H3K9me. Thus, in Clr4 chromodomain mutants catalytically proficient but incapable of binding H3K9me (20, 24), or in mutants lacking the HP1 homolog Swi6 (19, 24, 25), H3K9me heterochromatin is restricted to cenH. At centromeres or when heterochromatin is induced by artificially tethering Clr4, the Clr4 chromodomain is also critical to the epigenetic heterochromatic state (23, 26, 27). For Swi6, the positive feedback might be exerted by interacting HDAC complexes (28–30) that foster H3K9 methylation (25, 31), and the Swi6 paralog Chp2 is likely to function in a similar way (28, 29, 32–35). These H3K9me/chromodomain feedbacks would be missing in organisms where heterochromatin relies exclusively on other modifications (36, 37).Following stochastic nucleation at cenH, it is not known whether heterochromatin propagates outward gradually as a front of modified nucleosomes or whether the propagation follows intermittent dynamics. The experiments monitoring the de novo establishment of heterochromatin in the wild-type mating-type region could not distinguish between the two possibilities (3). We reasoned that increasing system size would exacerbate the differences in outcome of various modes of propagation, allowing distinguishing between them. We thus expanded the size of the mating-type region incrementally to a total length of 31 kb and determined the effects on the profiles of establishment of heterochromatic silencing. The experiment revealed a stunning mode of heterochromatin propagation exhibiting an all-or-none dynamics even across large regions. The discovery of slower establishment for larger regions favors a scenario where heterochromatin formation is facilitated by distance-independent recruitment from the silenced state.     

Tryptophanemia is controlled by a tryptophan-sensing mechanism ubiquitinating tryptophan 2,3-dioxygenase

Maintaining stable tryptophan levels is required to control neuronal and immune activity. We report that tryptophan homeostasis is largely controlled by the stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme responsible for tryptophan catabolism. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding noncatalytic exosites, resulting in rapid catabolism of tryptophan. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers and unmasks a four–amino acid degron that triggers TDO polyubiquitination by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. The nonmetabolizable analog alpha-methyl-tryptophan stabilizes tetrameric TDO and thereby stably reduces tryptophanemia. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia as required for both neurological and immune homeostasis.

Blood levels of essential amino acids are remarkably constant despite large variations in diet supply, but the mechanisms ensuring amino acid homeostasis remain poorly understood (1). Systemic homeostasis is particularly important for tryptophan given its key roles as a neurotransmitter precursor and a regulator of immune responses (2–5). In humans, tryptophanemia is stably maintained around 60 ± 15 µM (mean ± SD) (6). Tryptophan catabolism involves dioxygenation leading to the production of kynurenine and derivatives (7, 8). This first and rate-limiting step can be catalyzed by two enzymes: TDO and indoleamine 2,3-dioxygenase (IDO1). Despite functional homology, these two enzymes differ in sequence, structure, expression, and physiological role. TDO (gene name TDO2) is a tetrameric enzyme expressed in the liver and responsible for degradation of excess dietary tryptophan (7, 9, 10). IDO1 is monomeric, only expressed in immune and inflammatory sites and mostly involved in immunoregulation (7, 11–13). Tryptophan catabolism by IDO1 can locally suppress T lymphocyte responses by depleting tryptophan and producing kynurenine. This immunosuppressive effect is exploited by tumors to resist immune rejection, and IDO1 inhibitors have been developed for cancer immunotherapy (3, 14). While IDO1 activity produces detectable levels of kynurenine in the blood, TDO does not as the kynurenine produced by TDO undergoes further degradation in the liver along the kynurenine pathway, leading to NAD and/or quinolinic acid (8). However, TDO activity is needed to control tryptophanemia. TDO-knockout (TDO-KO) mice and TDO-deficient humans have plasmatic tryptophan concentrations eight- to ninefold higher than wild-type mice or healthy humans (9, 15). As a result, TDO-KO mice better reject tumors and have higher levels of serotonin and other tryptophan metabolites in the brain, resulting in anxiolytic modulation and increased neurogenesis (9, 16). TDO is also expressed in some human tumors and may contribute to tumoral immune resistance (10, 16–18).     

Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus–induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify Opn5-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

Myopia (nearsightedness) in school-age children is generally axial myopia, which is the consequence of elongation of the eyeball along the visual axis. This shape change results in blurred vision but can also lead to severe complications including cataract, retinal detachment, myopic choroidal neovascularization, glaucoma, and even blindness (1–3). Despite the current worldwide pandemic of myopia, the mechanism of myopia onset is still not understood (4–8). One hypothesis that has earned a current consensus is the suggestion that a change in the lighting environment of modern society is the cause of myopia (9, 10). Consistent with this, outdoor activity has a protective effect on myopia development (9, 11, 12), though the main reason for this effect is still under debate (7, 12, 13). One explanation is that bright outdoor light can promote the synthesis and release of dopamine in the eye, a myopia-protective neuromodulator (14–16). Another suggestion is that the distinct wavelength composition of sunlight compared with fluorescent or LED (light-emitting diode) artificial lighting may influence myopia progression (9, 10). Animal studies have shown that different wavelengths of light can affect the development of myopia independent of intensity (17, 18). The effects appear to be distinct in different species: for chicks and guinea pigs, blue light showed a protective effect on experimentally induced myopia, while red light had the opposite effect (18–22). For tree shrews and rhesus monkeys, red light is protective, and blue light causes dysregulation of eye growth (23–25).It has been shown that visible violet light (VL) has a protective effect on myopia development in mice, in chick, and in human (10, 26, 27). According to Commission Internationale de l’Eclairage (International Commission on Illumination), VL has the shortest wavelength of visible light (360 to 400 nm). These wavelengths are abundant in outside sunlight but can only rarely be detected inside buildings. This is because the ultraviolet (UV)-protective coating on windows blocks all light below 400 nm and because almost no VL is emitted by artificial light sources (10). Thus, we hypothesized that the lack of VL in modern society is one reason for the myopia boom (9, 10, 26).In this study, we combine a newly developed lens-induced myopia (LIM) model with genetic manipulations to investigate myopia pathways in mice (28, 29). Our data confirm (10, 26) that visible VL is protective but further show that delivery of VL only in the evening is sufficient for the protective effect. In addition, we show that the protective effect of VL on myopia induction requires OPN5 (neuropsin) within the retina. The absence of retinal Opn5 prevents lens-induced, VL-dependent thickening of the choroid, a response thought to play a key role in adjusting the size of the eyeball in both human and animal myopia models (30–33). This report thus identifies a cell type, the Opn5 retinal ganglion cell (RGC), as playing a key role in emmetropization. The requirement for OPN5 also explains why VL has a protective effect on myopia development.     

Pharmacological inhibition of PI5P4Kα/β disrupts cell energy metabolism and selectively kills p53-null tumor cells

Most human cancer cells harbor loss-of-function mutations in the p53 tumor suppressor gene. Genetic experiments have shown that phosphatidylinositol 5-phosphate 4-kinase α and β (PI5P4Kα and PI5P4Kβ) are essential for the development of late-onset tumors in mice with germline p53 deletion, but the mechanism underlying this acquired dependence remains unclear. PI5P4K has been previously implicated in metabolic regulation. Here, we show that inhibition of PI5P4Kα/β kinase activity by a potent and selective small-molecule probe disrupts cell energy homeostasis, causing AMPK activation and mTORC1 inhibition in a variety of cell types. Feedback through the S6K/insulin receptor substrate (IRS) loop contributes to insulin hypersensitivity and enhanced PI3K signaling in terminally differentiated myotubes. Most significantly, the energy stress induced by PI5P4Kαβ inhibition is selectively toxic toward p53-null tumor cells. The chemical probe, and the structural basis for its exquisite specificity, provide a promising platform for further development, which may lead to a novel class of diabetes and cancer drugs.

There are two synthetic routes for phosphatidylinositol 4,5-bisphosphate, or PI(4,5)P₂, a versatile phospholipid with both structural and signaling functions in most eukaryotic cells (1 –3). The bulk of PI(4,5)P₂ is found at the inner leaflet of the plasma membrane and is synthesized from phosphatidylinositol 4-phosphate, or PI(4)P, by type 1 phosphatidylinositol phosphate kinase PI4P5K (4, 5). A smaller fraction of PI(4,5)P₂ is generated from the much rarer phosphatidylinositol 5-phosphate, or PI(5)P, through the activity of type 2 phosphatidylinositol phosphate kinase PI5P4K (6, 7). Although PI5P4K is as abundantly expressed as PI4P5K (8), its function is less well understood (9). It has been proposed that PI5P4K may play a role in suppressing PI(5)P, which is often elevated by stress (10, 11), or produce local pools of PI(4,5)P₂ at subcellular compartments such as Golgi and nucleus (12).Higher animals have three PI5P4K isoforms, α, β, and γ, which are encoded by three different genes, PIP4K2A, PIP4K2B, and PIP4K2C. The three isoforms differ, at least in vitro, significantly in enzymatic activity: PI5P4Kα is two orders of magnitude more active than PI5P4Kβ, while PI5P4K-γ has very little activity (13). PI5P4Ks are dimeric proteins (14), and the possibility that they can form heterodimers may have important functional implications, especially for the lesser active isoforms (15, 16). PI5P4Kβ is the only isoform that preferentially localizes to the nucleus (17).Genetic studies have implicated PI5P4Kβ in metabolic regulation (18, 19). Mice with both PIP4K2B genes inactivated manifest hypersensitivity to insulin stimulation (adult males are also leaner). Although this is consistent with the observation that PI(5)P levels, which can be manipulated by overexpressing PI5P4K or a bacterial phosphatase that robustly produces PI(5)P from PI(4,5)P₂, correlate positively with PI3K/Akt signaling, the underlying molecular mechanisms remain undefined (20). Both male and female PIP4K2B ^−/− mice are mildly growth retarded. Inactivation of the only PI5P4K isoform in Drosophila also produced small and developmentally delayed animals (21). These phenotypes may be related to suppressed TOR signaling (22, 23), but again, the underlying mechanism is unclear since TORC1 is downstream of, and positively regulated by, PI3K/Akt. Knocking out the enzymatically more active PI5P4Kα, in contrast, did not produce any overt metabolic or developmental phenotypes (19).Malignant transformation is associated with profound changes in cell metabolism (24, 25). Although metabolic reprograming generally benefits tumor cells by increasing energy and material supplies, it can also, counterintuitively, generate unique dependencies (26, 27). Loss of p53, a tumor suppressor that is mutated in most human cancers, has been shown to render cells more susceptible to nutrient stress (28, 29) and to the antidiabetic drug metformin (30, 31). Although TP53 ^−/− and PIP4K2B ^−/− mice are themselves viable, combining the two is embryonically lethal (19). Knocking out three copies of PI5P4K (PIP4K2A ^−/− PIP4K2B ^+/− ) greatly reduces tumor formation and cancer-related death in TP53 ^−/− animals (19). The synthetic lethal interaction between p53 and PI5P4Kα/β was thought to result from suppressed glycolysis and increased reactive oxygen species (19), although how the lipid kinases impact glucose metabolism remains uncertain.Given the interest in the physiological function of this alternative synthetic route for PI(4,5)P₂, and the potential of PI5P4K inactivation in treating type 2 diabetes and cancer, several attempts have been made to identify chemical probes that target various PI5P4K isoforms, which yielded compounds with micromolar affinity and unknown selectivity (32 –35). Here, we report the development of a class of PI5P4Kα/β inhibitors that have much improved potency and better-defined selectivity. Using the chemical probe, we show that transient inhibition of the lipid kinases alters cell energy metabolism and induces different responses in muscle and cancer cells.     

Macrophage LC3-associated phagocytosis is an immune defense against Streptococcus pneumoniae that diminishes with host aging

Streptococcus pneumoniae is a leading cause of pneumonia and invasive disease, particularly, in the elderly. S. pneumoniae lung infection of aged mice is associated with high bacterial burdens and detrimental inflammatory responses. Macrophages can clear microorganisms and modulate inflammation through two distinct lysosomal trafficking pathways that involve 1A/1B-light chain 3 (LC3)-marked organelles, canonical autophagy, and LC3-associated phagocytosis (LAP). The S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers an autophagic response in nonphagocytic cells, but the role of LAP in macrophage defense against S. pneumoniae or in age-related susceptibility to infection is unexplored. We found that infection of murine bone-marrow-derived macrophages (BMDMs) by PLY-producing S. pneumoniae triggered Atg5- and Atg7-dependent recruitment of LC3 to S. pneumoniae-containing vesicles. The association of LC3 with S. pneumoniae-containing phagosomes required components specific for LAP, such as Rubicon and the NADPH oxidase, but not factors, such as Ulk1, FIP200, or Atg14, required specifically for canonical autophagy. In addition, S. pneumoniae was sequestered within single-membrane compartments indicative of LAP. Importantly, compared to BMDMs from young (2-mo-old) mice, BMDMs from aged (20- to 22-mo-old) mice infected with S. pneumoniae were not only deficient in LAP and bacterial killing, but also produced higher levels of proinflammatory cytokines. Inhibition of LAP enhanced S. pneumoniae survival and cytokine responses in BMDMs from young but not aged mice. Thus, LAP is an important innate immune defense employed by BMDMs to control S. pneumoniae infection and concomitant inflammation, one that diminishes with age and may contribute to age-related susceptibility to this important pathogen.

Streptococcus pneumoniae (pneumococcus) commonly colonizes the nasopharynx asymptomatically but is also capable of infecting the lower respiratory tract to cause pneumonia and spreading to the bloodstream to cause septicemia and meningitis (1). Susceptibility to pneumonia and invasive disease caused by S. pneumoniae is remarkably higher in individuals aged 65 and over, leading to high rates of mortality and morbidity in the elderly population (1, 2). In countries, such as the United States and Japan, deaths due to pneumococcal pneumonia have been on the rise in parallel with the rapid growth in the elderly population (3, 4).A hallmark of pneumococcal pneumonia is a rapid and exuberant response by immune cells, such as neutrophils and macrophages. This innate immune response to S. pneumoniae lung infection is critical for pathogen clearance and the control of disease (5–7). Deficiencies in the number or function of innate phagocytic cells, such as neutropenia (8) or macrophage phagocytic receptor defects (9–12), lead to diminished pneumococcal clearance and increased risk of invasive pneumococcal disease in both mouse models and humans. Phagocytic activity in alveolar macrophages is important during early responses to subclinical infections (13–15), and during moderate S. pneumoniae lung infection, newly generated monocytes eggress from the bone marrow and migrate into the lungs, differentiating into monocyte-derived alveolar macrophages (16). In addition to directly eliminating the invading microbe, macrophages secrete key cytokines, such as tumor necrosis factor (TNF), interleukin-1β (IL-1β), and interleukin-6 (IL-6), that regulate effector cell functions and pulmonary inflammation (17–19).Although an innate immune response is critical for pathogen clearance, poorly controlled inflammation can lead to tissue damage and mortality (20, 21). For example, in murine models, neutrophilic infiltration can enhance pulmonary damage and disrupt epithelial barrier function, leading to bacteremia and mortality (22–25). Macrophages are critical not only in regulating the early inflammatory response, but are also crucial for curtailing inflammation during the resolution phase of infection to limit tissue damage and promote healing (26, 27).Elderly individuals have higher baseline and induced levels of inflammation, a phenomenon termed inflammaging (28), that contributes to many age-associated pathological conditions, including increased susceptibility to a variety of infectious diseases, such as S. pneumoniae infection (7, 28–30). S. pneumoniae-induced inflammation, characterized by increased levels of chemokines, proinflammatory cytokines, and decreased anti-inflammatory cytokines, such as IL-10, is enhanced in the elderly (29, 31) as well as in aged mice (32, 33) and correlates with ineffective immune responses. Age-related chronic exposure to TNF-α, for instance, dampens macrophage-mediated S. pneumoniae clearance during lung infection (34), and NLR family pyrin domain containing 3 inflammasome activation in macrophages diminishes upon aging in mice (35). However, the age-related changes in macrophage effector functions leading to diminished clearance of S. pneumoniae are incompletely understood.One important means of macrophage-mediated pathogen clearance is1A/1B-light chain-3 (LC3)-associated phagocytosis (LAP), a process by which cells target phagocytosed extracellular particles for efficient degradation (36–38). LAP combines the molecular machineries of phagocytosis and autophagy, resulting in the conjugation of the autophagic marker, the microtubule-associated protein LC3, to phosphatidylethanolamine on the phagosomal membrane, generating so-called “LAPosomes” that undergo facilitated fusion with lysosomes (38, 39). Canonical autophagy targets cytoplasmic components, such as damaged subcellular organelles and intracellular microbes for sequestration into double-membrane autophagic vesicles (40, 41). In contrast, LAPosomes retain the single-membrane nature of phagosomes, and their formation requires overlapping but nonidentical genes compared to canonical autophagy (42). In addition to enabling efficient degradation of phagocytosed bacteria, LAP also plays important immune regulatory roles, such as in curtailing proinflammatory cytokine production during the subsequent innate immune response (39, 43). Indeed, the LAP-mediated microbial defense and immunomodulatory functions work together to limit tissue damage and restore homeostasis (38).S. pneumoniae triggers canonical autophagy in epithelial cells and fibroblasts, and bacteria can be found in double-membrane vacuoles whose formation is dependent on autophagic machinery (44). Many bacterial pathogens that induce autophagy produce pore-forming toxins, which can damage endosomal membranes, thus, recruiting autophagic machinery to engulf injured organelles (45). Pneumococcus-induced autophagy is dependent on the cholesterol-dependent pore-forming toxin pneumolysin (PLY), which triggers the autophagic delivery of S. pneumoniae to lysosomes and results in bacterial killing (44, 46). Recently, a kinetic examination of S. pneumoniae-targeting autophagy in fibroblasts demonstrated that canonical autophagy was preceded by early and rapid PLY-dependent LAP (47). However, the requirements for this process were somewhat different from LAP in macrophages, and the pneumococcus-containing LAPosomes did not promote bacterial clearance but required subsequent transition to canonical autophagy to reduce bacterial numbers (46, 47).In the current study, we found that S. pneumoniae infection of murine bone-marrow-derived macrophages (BMDMs) induces LAP in a PLY-dependent manners and that age-related defects in BMDM LAP contributed to diminished bactericidal activity and enhanced proinflammatory cytokine production. Our results suggest that PLY-induced LAP promotes bacterial clearance, and age-associated dysregulation of this process may contribute to enhanced bacterial survival, poorly regulated inflammation, and increased susceptibility to invasive pneumococcal disease.