THE IMMUNE RESPONSE AGAINST INTRACELLULAR BACTERIA

Examples of intracellular bacteria

Cooperation of CD4+ and CD8+ T cells in defense against intracellular microbes Intracellular bacteria such as L. monocytogenes are phagocytosed by macrophages and may survive in phagosomes and escape into the cytoplasm. CD4+ T cells respond to class II MHC–associated peptide antigens derived from the intravesicular bacteria. These T cells produce IFN-γ, which activates macrophages to destroy the microbes in phagosomes. CD8+ T cells respond to class I–associated peptides derived from cytosolic antigens and kill the infected cells. Cellular and Molecular Immunology, 7th ed., 2014 Elservier

Intracellular replication of Listeria monocytogenes a | L. monocytogenes induces its entry into a phagocyte. b | Bacteria are internalized in a vacuole (also known as a phagosome). c,d | The membrane of the vacuole is disrupted by the secretion of two phospholipases, PlcA and PlcB, and the pore-forming toxin listeriolysin O. Bacteria are released into the cytoplasm, where they multiply and start to polymerize actin, as observed by the presence of the characteristic actin tails. e | Actin polymerization allows bacteria to pass into a neighbouring cell by forming protrusions in the plasma membrane. f | On entry into the neighbouring cell, bacteria are present in a double-membraned vacuole, from which they can escape to perpetuate the cycle. F-actin, filamentous actin.

Cell-mediated immunity to Listeria monocytogenes Immunity to L. monocytogenes is measured by inhibition of bacterial growth in the spleens of animals inoculated with a known dose of viable bacteria. Such immunity can be transferred to normal mice by T lymphocytes (A) but not by serum (B) from syngeneic mice previously immunized with killed or low doses of L. monocytogenes. In an in vitro assay of cell-mediated immunity, the bacteria are actually killed by activated macrophages and not by T cells (C). Cellular and Molecular Immunology, 7th ed., 2014 Elservier

Immune responses to Listeria monocytogenes MHC I MHC II IL-12 Lysis of macrophage NK cell CD8+ cytotoxic T cell CD4+ Th1 cell IFNγ Macrophage activation IFN-γ secreted by NK cells activates macrophages to destroy phagocytosed microbes. This IFN-γ–dependent NK cell–macrophage reaction can control an infection with intracellular bacteria such as Listeria monocytogenes for several days or weeks and thus allow time for T cell–mediated immunity to develop and eradicate the infection Escape of bacteria to the cytoplasm listeriolysin Killing of bacteria in the macrophage

Innate and adaptive immunity to intracellular bacteria The innate immune response to intracellular bacteria consists of phagocytes and NK cells, interactions among which are mediated by cytokines (IL-12 and IFN-γ). The typical adaptive immune response to these microbes is cell-mediated immunity, in which T cells activate phagocytes to eliminate the microbes. Innate immunity may control bacterial growth, but elimination of the bacteria requires adaptive immunity. These principles are based largely on analysis of Listeria monocytogenes infection in mice; the numbers of viable bacteria shown on the y-axis are relative values of bacterial colonies that can be grown from the tissues of infected mice. (Data from Unanue ER. Studies in listeriosis show the strong symbiosis between the innate cellular system and the T-cell response. Immunological Reviews 158: 11-25, 1997.) Cellular and Molecular Immunology, 7th ed., 2014 Elservier

Infection by Mycobacterium tuberculosis Infection is initiated by the inhalation of aerosol droplets that contain bacteria. The initial stages of infection are characterized by innate immune responses that involve the recruitment of inflammatory cells to the lung. Following bacterial dissemination to the draining lymph node, dendritic cell presentation of bacterial antigens leads to T cell priming and triggers an expansion of antigen-specific T cells, which are recruited to the lung. The recruitment of T cells, B cells, activated macrophages and other leukocytes leads to the establishment of granulomas, which can contain Mycobacterium tuberculosis. Most infected individuals will remain in a 'latent' state of infection, in which no clinical symptoms are present. A small percentage of these people will eventually progress and develop active disease, which can lead to the release of M. tuberculosis from granulomas that have eroded into the airways. When individuals with active tuberculosis (TB) cough, they can generate infectious droplets that transmit the infection.

Mutual activation of macrophages and effector lymphocytes in the immune response to intracellular bacterial infections In the innate immune response (left panel), macrophages are activated by the IFN-γ made by NK cells and in turn produce the cytokine IL-12. This binds to IL-12 receptors on the NK cells, inducing further secretion of IFN-γ and maintenance of macrophage activation. In the adaptive immune response (right panel), IL-12 secreted by macrophages acts on TH1 cells, inducing their differentiation into IFN-γ-secreting TH1 cells. CD8 cytotoxic T cells (CTLs) also respond to the IL-12 made by the macrophage and they too produce IFN-γ. In immunodeficient patients lacking the IL-12 receptor or the IFN-γ receptor, this cycle of mutual activation cannot proceed, so infection persists.

Effector functions of macrophages Macrophages are activated by microbial products such as LPS and by NK cell–derived IFN-γ. The process of macrophage activation leads to the activation of transcription factors, the transcription of various genes, and the synthesis of proteins that mediate the functions of these cells. In adaptive cell-mediated immunity, macrophages are activated by stimuli from T lymphocytes (CD40 ligand and IFN-γ) and respond in essentially the same way. Cellular and Molecular Immunology, 7th ed., 2014 Elservier

Infected macrophages send signals that recruit nearby lymphocytes

Uninfected macrophages and lymphocytes surround the infected cells M. tuberculosis is capable of surviving within macrophages because components of its cell wall inhibit the fusion of phagocytic vacuoles with lysosomes. Continuing T cell activation leads to the formation of granulomas, which attempt to wall off the bacteria and are often associated with central necrosis, called caseous necrosis, which is caused by macrophage products such as lysosomal enzymes and reactive oxygen species. Necrotizing granulomas and the fibrosis (scarring) that accompanies granulomatous inflammation are the principal causes of tissue injury and clinical disease in tuberculosis.

They encase the infected cells in a “cage” of “extracellular matrix” proteins where they can stay for years!

We are actually quite good at keeping TB at bay only one-third of exposed patients will become infected and only 3-5% develop TB in the first year Clin Microbiol Rev. 2003 July; 16(3): 463–496.

Granulomatous disease can become quite extensive Granulomatous disease can become quite extensive. Here are numerous confluent granulomas in a case of pulmonary tuberculosis Pulmonary granulomas. Granulomatous inflammation typically consists of epithelioid macrophages, giant cells, lymphocytes and fibroblasts. There may be some neutrophils.

Role of T cells and cytokines in determining the outcome of infections Naive CD4+ T lymphocytes may differentiate into TH1 cells, which activate phagocytes to kill ingested microbes, and TH2 cells, which inhibit this classical pathway of macrophage activation. The balance between these two T cell subsets may influence the outcome of infections, as illustrated by Mycobacterium leprae infections in humans. Cellular and Molecular Immunology, 7th ed., 2014 Elservier

Infection by mycobacterium leprae „Lepromatous” skin blebs Small lesion Tuberculoid Infectious bacteria Minimal response Vigorous T-cell response Suppressed Th1 response Local inflammation No efficient protection Balanced protection Granulomas Peripheral nerve damage SPECTRAL DISEASE DEPENDING ON THE IMMUNE RESPONSE Genetically/environmentally determined Th1/Th2

„Lepromatous” skin blebs Tuberculoid form

The immune response to intracellular bacteria INNATE IMMUNITY Phagocytosis – neutrophil granulocyte macrophage NK cells – direct activation of macrophages by IFNγ macrophage – mediated activation by IL - 12 ACQUIRED IMMUNITY Cell mediated – macrophage activation by CD4+ Th1 cells activation of CD8+ cytotoxic T cells Granuloma - isolation of bacteria, which resist elimination localized inflammatory response

Movie Allthree

Evasion of immune mechanisms by intracellular bacteria Inhibition of phagolysosome formation Mycobacterium tuberculosis Legionella pneumophilia Scavenging of reactive oxigen intermediates Mycobacterium leprae (phenolic glycolipid) Disruption of phagosome membrane, escape into cytoplasm Listeria monocytogenes (listeriolysin protein)

THE IMMUNE RESPONSE TO PARASITES

Unicellular protozoa Toxoplasma Plasmodium (malaria) Leishmania The principal innate immune response to protozoa is phagocytosis, but many of these parasites are resistant to phagocytic killing and may even replicate within macrophages.

The life cycle of Plasmodium Inoue SI et al. (2013) Front Immunol 4:258. Immune response Effector mechanism TH1 cells – secretion of cytokines Role of antibody? IFN-g, TNF activate macrophages, neutrophils to kill parasites Malaria

Life cycle of Leishmania Immune response Effector mechanism T cells produce IFN-g --> activation of phagocytes Phagocytes kill parasites living in endosomes Leishmania

Life cycle of Toxoplasma The acute phase of this infection lasts for less than around ten days. The parasite causes a very strong type-1 response focused on the interferon-gamma secreted by the T lymphocytes. This immune response limits the tissue extension of the parasite, ensuring the survival of the host, but, paradoxically, also aiding the survival of the parasite by converting it into a bradyzoite, an intracellular quiescent resistant form persisting in the muscle and brain tissues. Immune response Effector mechanism IFN-g, TNF activate macrophages, neutrophils to kill parasites Toxoplasma Strong TH1 response

Toxoplasma gondii, the „brain-hacker” parasite The genome of T. gondii encodes two aromatic hydroxylases that allow the parasite to synthesize dopamine. This may influence the behavior of seropositive indviduals (e.g. increased risk-taking). Recently, T. gondii infections have been correlated with many neuropsychiatric diseases: schizophrenia (38 large cohort studies, strong positive correlation); obsessive-compulsive disorder (7 large cohort studies, positive correlation) credit: DJP Feruson/University of Oxford

Multicellular parasites (helminths) Trichina worm - Trichinella spiralis Broad fish tapeworm - Diphyllobothrium latum They are too large to be ingested by phagocytes. Immune response Effector mechanism TH2 cells --> IL-4, IL-5 --> IgE, eosinophils Eosinophils kill IgE-coated parasites (form of ADCC)

Functions of Th2 cells CD4+ T cells that differentiate into TH2 cells secrete IL-4, IL-5, and IL-13. IL-4 (and IL-13) act on B cells to stimulate production of antibodies that bind to mast cells, such as IgE. Help for antibody production may be provided by Tfh cells that produce TH2 cytokines and reside in lymphoid organs, and not by classical TH2 cells. IL-4 is also an autocrine growth and differentiation cytokine for TH2 cells. IL-5 activates eosinophils, a response that is important for defense against helminthic infections. IL-4 and IL-13 are involved in immunity at mucosal barriers, induce an alternative pathway of macrophage activation, and inhibit classical TH1-mediated macrophage activation. Cellular and Molecular Immunology, 8th ed., 2015 Elservier

Responses to intestinal helminths CD4 T-cell responses to intestinal helminths usually polarize, becoming either a protective TH2 response (first four panels) or a pathological TH1 response (last two panels). TH2 responses lead to killing and expulsion of the parasite, whereas TH1 responses lead to persistent infection and chronic debilitating diseases of varying severity. MBP, major basic protein.

The role of T cell-mediated responses in defense against helminths Eosinophils are better at killing helminths than are other leukocytes; the TH2 response and IgE provide a mechanism for bringing eosinophils to helminths and activating the cells.

Life cycle of Schistosoma Schistosoma mansoni Delayed Type Hypersensitivity - DTH Fibrosis around the eggs in the liver Chronic inflammation – Fibrotic connective tissue Inhibits the venous circulation of the liver

Granuloma in the liver Schistosomiasis is a parasitic disease affecting more than 200 million people. Its major pathology is granulomatous inflammation, a cellular immune response to antigens secreted by schistosome ova.

Chronic schistosomiasis Eggs deposited in the intestinal veins are carried by portal blood flow to the liver inflammatory reaction results in periportal fibrosis and hepatic enlargement. Portal hypertension can develop and cause hepatosplenomegaly, ascites, and esophageal varices.

Activated eosinophils attack parasites Large parasites, such as worms or the schistosome larva (SL) shown here, cannot be ingested by phagocytes. However, they can be attacked by activated eosinophils (E) that are coated with anti-parasite IgE antibodies bound to FcεRI. When the eosinophils encounter the parasite, the parasite’s antigens will cross-link the IgE bound to FcεRI and activate the eosinophils to secrete the toxic and crippling contents of its granules directly on to the surface of the parasite.

Eosinophil granulocytes

Mast cells

Features that characterize a protective immune response to a helminth infection

Escape mechanisms of parasites Poor antigenicity Variations in surface structure – gene conversion Alternating expression (Trypanosoma) Privileged sites isolated from the immune system (cyst) Intracellular (Leishmania, Toxoplasma) Inhibition of phagosome and lysosome fusion (Toxoplasma) Antigen masking by bound self proteins Complement (DAF) like structures