Exam 3: The Induced Responses of Innate Immunity

Two strains of mice were infected with 5 $\times$ 10⁴ PFU of Influenza A virus, and the survival data shown in Figure were obtained. Next, both strains were infected again with Influenza A, and levels of type I interferons (IFN- $\alpha$ and IFN- $\beta$ ) were measured and found to be similar between the two strains. Likewise, cells from both strains expressed similar levels of the IFN- $\alpha$ / $\beta$ receptor (IFNAR). Which of the following proteins might be more highly expressed in strain B than in strain A following Influenza A infection?

Dendritic cells in the skin, known as Langerhans cells, express very high levels of the NOD-like receptor, NLRP3. Previous studies showed that treatment of these cells with the Staphylococcus aureus pore-forming toxin causes K⁺ efflux from the cells. To investigate whether this signal could induce IL-1 (an inflammatory cytokine) secretion by the cells, the following study was performed: The explanation for these results is:

The first pattern recognition receptor (PRR) important in innate immune responses was discovered in the fruit fly Drosophila melanogaster. Stimulation of this receptor, called Toll, induces:

In the sea urchin, a massive diversification of innate recognition receptors has occurred, resulting in the presence of over 200 TLR genes, over 200 NOD-like receptor genes, and over 200 scavenger receptor genes in the genome of these organisms. These receptors are unlikely to contribute to an enhanced innate immune response in sea urchins, because nearly all of these genes are pseuodgenes.

Immunodeficiency diseases occur when individuals have defects in leukocyte adhesion to inflamed endothelial cells, thereby impeding the extravasation of phagocytes into infected tissues. When neutrophils from one class of these patients were isolated and tested using in vitro assays for neutrophil-endothelial cell interactions and extravasation, it was found that the neutrophils could slowly roll along the endothelial vessel wall but were unable to arrest and migrate across the endothelium. The most likely protein deficient in these neutrophils is:

Many different viruses encode proteins that function to down-regulate MHC class I expression on host cells following infection with the virus. This immune evasion mechanism allows the virus to hide from CD8 T lymphocytes that normally detect virus-infected cells by using their T cell antigen receptor to recognize viral peptides bound to MHC class I proteins on the surface of the infected cell. To counteract this immune evasion strategy, NK cells have:

An infection in the skin, such as a pimple, often produces pus. The major component of pus is:

To identify genes encoding the receptors for the cytokines IL-2, IL-4, and IL-7, an siRNA screen is performed using purified T lymphocytes. To identify siRNAs that knock-down cytokine receptor expression, the T cells have been transfected with a construct that produces green fluorescent protein (GFP) when any one of these three cytokines is used to stimulate the cells. When the screen is completed, several different siRNAs have been identified that substantially reduce the T cells ability to respond to these cytokines as shown in Figure . A correct statement regarding these data is:

The acute phase response contributes to infection control by producing molecules that promote pathogen opsonization and complement activation. This response is only induced by direct action of microbial components on hepatocytes in the liver.

Streptococcus pyogenes is a Group A Streptococcal (GAS) bacterium that causes a variety of diseases, depending on the tissue that is infected. Most frequently, this bacterium causes strep throat and localized infections in the skin. However, on occasion, the bacteria spread to the blood, and can a cause life-threatening infection that has a mortality rate of ~25%. Studies in mouse models have shown that the recruitment of neutrophils to the site of infection is essential in the efficient elimination of a Group A strep infection. To determine the innate immune pathways involved in protection against GAS, mice lacking the adapter protein MyD88 were tested for their response to GAS compared with wild-type (C57BL/6) control mice. For these studies, mice were infected with 10⁴ PFU of GAS subcutaneously (under the skin). The results are shown in Figure. To assess the magnitude of the increased susceptibility of MyD88^-/- mice to GAS, mice were tested with increasing doses of bacteria, and the LD₅₀ (dose required to generate a lethal infection in 50% of the mice) was calculated. The conclusion of this analysis was an LD₅₀ of 2 $\times$ 10⁶ PFU for the wild-type mice, and <1 $\times$ 10⁴ for the MyD88^-/- mice. In addition, when mice were infected with 5 $\times$ 10⁷ PFU of GAS subcutaneously, as shown in Figure panel A, all the mice eventually succumbed to the infection, but the average survival time of the wild-type mice was significantly longer than for the MyD88^-/- mice. In addition, measurements of bacterial counts in the blood within the first day post-infection (panel B) confirmed the poor innate immune response of the MyD88^-/- mice. However, when various knockouts of individual TLRs were tested, none of them showed increased susceptibility to GAS of a magnitude similar to the MyD88^-/^- mice; similarly, double knockouts of several TLRs in various combinations also failed to recapitulate the severe defect in innate immune response observed in the absence of MyD88. a) Propose an explanation for the importance of MyD88 in the innate immune response to GAS. b) If TLRs are not required for the innate immune response to GAS, what other sensor of bacterial components is a good candidate to induce the transcription of pro-inflammatory cytokine genes? To investigate the innate immune response to GAS further, macrophages were isolated from mice, and cultured together with live GAS bacteria. In addition to macrophages from wild-type mice, macrophages were also isolated from MyD88^-/-, RIP2^-/-, TAK1^-/- and ASC^-/- mice. After 18 hours of incubation, the supernatants from these cultures were test for IL-1 $\beta$ and TNF- $\alpha$ concentrations. The results are shown in Figure C.signaling; RIP2 for NOD receptor signaling; TAK1 for TLR and NOD receptor signaling; ASC for NLRP3 signaling to activate Caspase-1) c) Based on these data, what can you conclude about the function of MyD88 in the innate response to GAS? d) On the graphs in Figure , draw the expected results for IL-1β and TNF- $\alpha$ mRNA levels in these macrophages. The results from wild-type macrophages are shown for reference.

NK cells can be activated following recognition of a virus-infected cell, if that cell has down-regulated expression of MHC class I proteins on its surface. However, NK cells can also recognize infected cells or tumor cells, even if they still express MHC class I proteins. In this latter case, activating receptors on NK cells are recognizing:

In healthy adults, neutrophils represent approximately half of their white blood cells. During a bacterial infection, this number often rises to >80%. One factor contributing to this rise is:

The NBT (Nitro Blue Tetrazolium) test is used to diagnose the genetic disorder Chronic Granulomatous Disease (CGD). To perform this test, peripheral blood cells from the patient are stimulated with bacterial extracts, and then incubated with the NBT compound. Normal neutrophils turn blue in this test due to cleavage of the compound, while patient neutrophils remain uncolored, as shown in Figure. Name a neutrophil receptor that is likely to be stimulated by the bacterial extract in this assay, and describe how this receptor regulates the activity of the enzyme that cleaves NBT.

The inflammatory response is characterized by four classic symptoms: heat, redness, pain, and swelling. In some instances, this response can be triggered by stimuli that are non-infectious such as asbestos, a process known as 'sterile inflammation.' When exposure to the stimulating trigger is persistent, a state of chronic inflammation can result. This process is likely to be detrimental to the health of the host.

Stimulation of the nucleic acid sensing TLRs that reside in endosomal membranes induces the production of a different cytokine response than is produced by stimulation of the plasma membrane TLRs. In part, this distinction is based on the different adapter proteins used by the nucleic acid sensing TLRs, leading to the activation of IRF factors. The cytokine response following stimulation of nucleic acid-sensing TLRs is characterized by production of:

Chemokines such as CXCL8 have a key role in the rapid recruitment of neutrophils to the site in the tissue containing the focus of an infection. In this response, CXCL8 has two different functions. In addition to inducing integrin activation on the neutrophil, CXCL8 also functions to:

Most normal tissues contain resident macrophages, and connective tissue sites in the gastrointestinal tract and the lung contain large numbers of these cells. Yet the blood also contains a high number of circulating 'classical' monocytes that can differentiate into macrophages after entering tissues. These circulating monocytes function to:

Signaling through the Drosophila Toll pathway is initiated when pathogen recognition receptors (PRRs) bind to microbial products, such as bacterial peptidoglycan. Aspects of this pathway share similarity to the mammalian complement cascade as well as to the innate recognition system based on TLRs. One feature of Toll signaling that resembles the complement pathway is:

Chemokines are small chemoattractant molecules made by epithelial cells, tissue macrophages, and endothelial cells in response to infection or injury. They differ slightly in sequence and structure based on the cells that secrete them, but all of them act to recruit both monocytes and neutrophils from the blood.