**2. Epidemiology**

#### **2.1. Epidemiology of HIV pandemic**

HIV infection is a viral disease that affects host immune system and makes the host strongly susceptible to opportunistic infections. It spreads among human population via sexual contact, sharing needles during drug injection, transfusion of contaminated blood products, or during births from HIV-infected women.

Since the late 20th century, HIV infection has become a global pandemic and a major challenge for public health authorities at the national and international levels. At the end of 2013, 35 million people were living with HIV worldwide [2]. During the same year, approximately 2.1 million people were newly infected with HIV. Sub-Saharan Africa is the most affected region in the world—almost 70% of the prevalent and the new HIV-positive cases are diagnosed there. The socio-economic status of the countries in the core of HIV pandemic determines the low number of infected people currently on adequate antiretroviral therapy (only 36% of overall cases).

#### **2.2. Epidemiology of tuberculosis**

Tuberculosis (TB) is an infectious disease caused by bacterial species grouped in *Mycobacterium tuberculosis* complex—MBTC (*M. bovis*, *M. africanum*, *M. canetti*, *M. caprae*, *M. microti*, *M. pinnipedii).* Among these mycobacterial species, the most important and major cause of human tuberculosis is *Mycobacterium tuberculosis*. It is a widespread microorganism that readily colonizes and infects humans. Primary infection represents clinical manifestation in only 10% of infected individuals, while in the remaining 90%, *M. tuberculosis* stays in latent form without showing any obvious clinical signs—latent (dormant) tuberculosis [3].

The World Health Organization (WHO) recognizes TB as "a global public health emergency" and "one of the world's deadliest communicable diseases" [1]. At least one-third of the world population (approx. 2 billion people) is currently infected with latent mycobacteria and has a risk, although low (5–10%), to develop an active disease during the life course [1]. In 2013, 9 million new TB cases occurred worldwide (TB incidence: 126 cases per 100,000 population) and the prevalent cases were 11 million (TB prevalence: 159 cases per 100,000 population). Despite the efforts at the international, national, and regional level and the significant progress in the detection and management of TB infection, still the incidence, prevalence, and mortality rate of TB are unacceptably high.

#### **2.3. Epidemiology of TB/HIV coinfection**

individual (coinfection), they act in synergy to deteriorate the defense mechanisms of the host's

In this chapter, we will review current knowledge of innate and adaptive immune responses against TB and HIV infections. We will discuss in detail the immunological basis behind the

HIV infection is a viral disease that affects host immune system and makes the host strongly susceptible to opportunistic infections. It spreads among human population via sexual contact, sharing needles during drug injection, transfusion of contaminated blood products, or during

Since the late 20th century, HIV infection has become a global pandemic and a major challenge for public health authorities at the national and international levels. At the end of 2013, 35 million people were living with HIV worldwide [2]. During the same year, approximately 2.1 million people were newly infected with HIV. Sub-Saharan Africa is the most affected region in the world—almost 70% of the prevalent and the new HIV-positive cases are diagnosed there. The socio-economic status of the countries in the core of HIV pandemic determines the low number of infected people currently on adequate antiretroviral therapy (only 36% of overall

Tuberculosis (TB) is an infectious disease caused by bacterial species grouped in *Mycobacterium tuberculosis* complex—MBTC (*M. bovis*, *M. africanum*, *M. canetti*, *M. caprae*, *M. microti*, *M. pinnipedii).* Among these mycobacterial species, the most important and major cause of human tuberculosis is *Mycobacterium tuberculosis*. It is a widespread microorganism that readily colonizes and infects humans. Primary infection represents clinical manifestation in only 10% of infected individuals, while in the remaining 90%, *M. tuberculosis* stays in latent form without

The World Health Organization (WHO) recognizes TB as "a global public health emergency" and "one of the world's deadliest communicable diseases" [1]. At least one-third of the world population (approx. 2 billion people) is currently infected with latent mycobacteria and has a risk, although low (5–10%), to develop an active disease during the life course [1]. In 2013, 9 million new TB cases occurred worldwide (TB incidence: 126 cases per 100,000 population) and the prevalent cases were 11 million (TB prevalence: 159 cases per 100,000 population). Despite the efforts at the international, national, and regional level and the significant progress in the detection and management of TB infection, still the incidence, prevalence, and mortality

showing any obvious clinical signs—latent (dormant) tuberculosis [3].

immunity and to accelerate the fatal scenario.

dual threat of TB/HIV coinfection.

56 Immunopathology and Immunomodulation

**2.1. Epidemiology of HIV pandemic**

births from HIV-infected women.

**2.2. Epidemiology of tuberculosis**

rate of TB are unacceptably high.

**2. Epidemiology**

cases).

People living with HIV have 26–31 times higher risk to develop active TB than the normal population [1]. Both diseases are epidemiologically and biologically connected—TB is the leading cause of death in HIV-positive patients and in turn HIV infection exacerbates the gravity of TB. Due to the global circulation of HIV, the incidence of TB —an infection previously thought to be almost eliminated at least in developed countries—is still high in both developing and high-income countries. HIV disrupts the host immune system through significant depletion of CD4+ lymphocytes, a mechanism that readily facilitates the reactivation of latent TB to an active disease. Therefore, HIV is the strongest risk factor for the development of new or the reactivation of dormant TB disease, including multidrug-resistant TB, an infection resistant to at least two of the first choice drugs for TB treatment, isoniazid and rifampicin. HIV infection predisposes TB-infected patients to antibiotic (rifampicin) resistance through gastrointestinal malabsorption of TB medications [4]. On the other hand, *M. tuberculosis* enhances HIV replication [5] and decreases the CD4+ T cell counts in HIV-positive patients [6].

As one-third of the total world population is currently infected with latent *M. tuberculosis* [1] and almost 1% of the adult population is living with HIV, it is not surprising that 1.1 million (13%) of the 9 million people who developed TB in 2013 were at the same time HIV-positive and 360,000 of the overall 1.5 million TB deaths for 2013 were HIV-positive. Most of them (78%) were living in low-income economies in Sub-Saharan Africa, a region where 50–80% of TB patients have HIV coinfection[1].

WHO recommends regular screening of TB for all people living with HIV at every visit to a health care specialist [7]. WHO also recommends that routine HIV testing should be offered to all patients with presumptive and diagnosed TB, as well as to partners of known HIVpositive TB patients.

In addition to the increase in TB incidence, the worldwide HIV pandemic changed the average age of TB-infected people. In contrast to normal populations where TB is most prevalent among elderly people, in regions with significant number of HIV-positive cases, the most affected age by TB is the reproductive age (20–45 years). This leads to the consecutive increase in the number of TB-infected children [8].

## **3. Immunology of tuberculosis**

#### **3.1. The first date**

Primary infection with *M. tuberculosis* occurs after inhalation of aerosolized infectious nuclei containing mycobacterial cells [9]. Any person with active TB can transmit the pathogen through coughing or sneezing, infecting 2–10 healthy individuals. Tubercle bacilli survive in the air for a short time (few hours), but the infective dose is relatively low (only 1–10 living cells). Infectious nuclei bigger than 10 µm gravitate in the nasal conchae and the nasopharynx. Those measuring between 5 and 10 µm enter the lower respiratory tract and mucociliary escalator eliminates them. Only infectious droplets smaller than 5 µm persist in the distal lung alveoli and can cause infection.

In most cases, the non-immune mechanisms do not allow the development of the infectious process. If infection occurs, the immune system in the lungs activates the first-line innate defense and then, several weeks later, an adaptive anti-mycobacterial immune response is activated. The strong immune response against *M. tuberculosis* works in two warring interests —on one hand, it restricts *M. tuberculosis* dissemination outside the initial infection place, and on the other hand, it supports its survival and silent presence for years in healthy individuals. The human immune system succeeds to completely eliminate *M. tuberculosis* in only 10% of infected cases, while in the remaining 90%, it fulfills in different degrees and controls the infection by turning it into the latent state [3].

#### **3.2. Innate immunity**

The early stages of TB infection consist of inhalation of tubercle bacilli and initial encounter between the immune system and the pathogen. Alveolar macrophages and sometimes nonprofessional phagocytic cells (alveolar epithelial cells) are the first to recognize *M. tuberculosis* cells [10, 11, 12]. Still, unknown intrinsic virulent features of *M. tuberculosis* strains and individual host immune differences are crucial for the fate of tubercle bacilli in the early days of infection. The most favorable outcome is the definitive destruction of *M. tuberculosis* by non-specific defense mechanisms in the macrophages. In this case, adaptive immunity does not develop and participate in the protection.

If bacteria survive bactericidal macrophage action, they can multiply intracellularly to destroy the infected macrophage and to release attractants for monocyte and dendritic cells accumu‐ lation. The attracted monocytes will differentiate into macrophages that in turn can recognize new *M. tuberculosis* cells to increase the population size of infected cells in the lung and sometimes in extra pulmonary locations. All these immune cells readily engulf *M. tuberculo‐ sis* cells but are unable to completely destroy them. Thus, the number of *M. tuberculosis* in the place of infection progressively increases.

Macrophages and dendritic cells bind *M. tuberculosis* cells via different receptors: toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD-) like receptors (NLRs), mannose receptors (CD207), dendritic cell-specific intercellular adhesion molecule grabbing nonintegrins (DC-SIGN), Dectin-1 receptors, complement receptors, and others [3]. Among TLRs, the most important for pulmonary TB cases are TLR2, TLR4, and TLR9 [10–12]. TLR2 recognizes tubercle polysaccharides and via binding with TLR1 can identify tubercle cell wall 19 and 27 kDa lipoproteins—important *M. tuberculosis* cell surface ligands. Furthermore, bacterial DNA released after bacterial destruction in lisosomes activates TLR9.

The interaction between *M. tuberculosis* and TLRs induces a signal proinflammatory cascade and provokes secretion of cellular signals—TNF-alpha, IL-1, IL-6, IL-12, IL-18, IL-15, IL-23, IFN-gamma, and chemokinases. The infected macrophages also release small molecules chemokines CCL, CCL3, CCL4, CCL5 (8–10 кDa)—to attract other blood monocytes and neutrophils [13], which after differentiation can directly display bactericidal action towards *M. tuberculosis*. Interleukins and chemokines serve both to attract other immune cells (lym‐ phocytes) and to activate them.

#### **3.3. Cell-mediated immunity**

escalator eliminates them. Only infectious droplets smaller than 5 µm persist in the distal lung

In most cases, the non-immune mechanisms do not allow the development of the infectious process. If infection occurs, the immune system in the lungs activates the first-line innate defense and then, several weeks later, an adaptive anti-mycobacterial immune response is activated. The strong immune response against *M. tuberculosis* works in two warring interests —on one hand, it restricts *M. tuberculosis* dissemination outside the initial infection place, and on the other hand, it supports its survival and silent presence for years in healthy individuals. The human immune system succeeds to completely eliminate *M. tuberculosis* in only 10% of infected cases, while in the remaining 90%, it fulfills in different degrees and controls the

The early stages of TB infection consist of inhalation of tubercle bacilli and initial encounter between the immune system and the pathogen. Alveolar macrophages and sometimes nonprofessional phagocytic cells (alveolar epithelial cells) are the first to recognize *M. tuberculosis* cells [10, 11, 12]. Still, unknown intrinsic virulent features of *M. tuberculosis* strains and individual host immune differences are crucial for the fate of tubercle bacilli in the early days of infection. The most favorable outcome is the definitive destruction of *M. tuberculosis* by non-specific defense mechanisms in the macrophages. In this case, adaptive immunity does

If bacteria survive bactericidal macrophage action, they can multiply intracellularly to destroy the infected macrophage and to release attractants for monocyte and dendritic cells accumu‐ lation. The attracted monocytes will differentiate into macrophages that in turn can recognize new *M. tuberculosis* cells to increase the population size of infected cells in the lung and sometimes in extra pulmonary locations. All these immune cells readily engulf *M. tuberculo‐ sis* cells but are unable to completely destroy them. Thus, the number of *M. tuberculosis* in the

Macrophages and dendritic cells bind *M. tuberculosis* cells via different receptors: toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD-) like receptors (NLRs), mannose receptors (CD207), dendritic cell-specific intercellular adhesion molecule grabbing nonintegrins (DC-SIGN), Dectin-1 receptors, complement receptors, and others [3]. Among TLRs, the most important for pulmonary TB cases are TLR2, TLR4, and TLR9 [10–12]. TLR2 recognizes tubercle polysaccharides and via binding with TLR1 can identify tubercle cell wall 19 and 27 kDa lipoproteins—important *M. tuberculosis* cell surface ligands. Furthermore,

The interaction between *M. tuberculosis* and TLRs induces a signal proinflammatory cascade and provokes secretion of cellular signals—TNF-alpha, IL-1, IL-6, IL-12, IL-18, IL-15, IL-23, IFN-gamma, and chemokinases. The infected macrophages also release small molecules chemokines CCL, CCL3, CCL4, CCL5 (8–10 кDa)—to attract other blood monocytes and neutrophils [13], which after differentiation can directly display bactericidal action towards

bacterial DNA released after bacterial destruction in lisosomes activates TLR9.

alveoli and can cause infection.

58 Immunopathology and Immunomodulation

**3.2. Innate immunity**

infection by turning it into the latent state [3].

not develop and participate in the protection.

place of infection progressively increases.

A fundamental characteristic of *M. tuberculosis* infection is the considerable delay in the onset of adaptive immunity, achieved by efficient control and management of the innate immunity; the host establishes an effective cell-mediated immune response several weeks (2–8) after the initial infection [3]. Nonetheless, the initiation of cell-mediated adaptive immunity, though delayed, is crucial for efficient control of further TB invasion.

The matured dendritic cells move to the regional lymph nodes where they initiate specific cell immune response by presenting the ingested mycobacterial antigens to the naïve T cells. Dendritic cells are a primary target for *M. tuberculosis* after aerosolic lung infection [14, 15]. They are professional antigen presenting cells (more effective than macrophages) and strong T cell activators. One of the mechanisms used by *M. tuberculosis* to delay the cell-mediated immunity is the efficient postponement of the movement of dendritic cells towards regional lymph nodes. In the lymph nodes, infected dendritic cells produce IL-12 [16] for the activation of NK cells and stimulation of INF-gamma release from T-lymphocytes [17]. Antigen present‐ ing dendritic cells prompts T cells to differentiate and to migrate, under the navigation of secreted adhesion molecules and chemokines, to the initial site of infection.

The concentration of macrophages, T-lymphocytes, dendritic cells, and other immune and lung cells (epithelial, giant multinuclear Langerhans' cells, plasma cells, neutrophils, fibro‐ blasts) in the place of infection is known as granuloma. The process of granuloma formation limits the spread of *M. tuberculosis* to other organs and restricts tissue's damage by separating the infected place [18]. But at the same time, granuloma microenvironment ensures the needed conditions for mycobacterial growth and multiplication.

Both human and animal granulomas contain giant multinuclear Langerhans' cells produced after the fusion of macrophages [18, 19]. In these cells, tubercle bacilli cannot multiply and survive successfully.

Neutrophils are important for early control of acute bacterial infection [20] as they immediately migrate to the place of mycobacterial infection and start bacterial phagocytosis [21]. Infected neutrophils produce IL-8 and TNF for activation of alveolar macrophages and limitation of the infection [22].

Both CD4+ and CD8+[23] contribute to the stimulation of macrophages and lysis of chronically infected macrophages (via INF-gamma action). The principal role in cell-mediated immunity against tuberculosis is played by CD4*+* T-lymphocytes. CD4+ express *a*/*b* T cell receptors to recognize mycobacterial antigens on the surface of antigen-presenting cells such as monocytes, macrophages, or dendritic cells [24]. Then, CD4+ lymphocytes efficiently induce apoptosis of macrophages [25] and stimulate the cytotoxic function of CD8+ [26].

CD8+ cells play an important role in IFN-gamma release, cell lysis and bacterial killing [27]. Some CD8+ lymphocytes may also recognize surface antigens presented by Class I MHC molecules—MHC class I-restricted cytotoxic T-lymphocytes (CTL). They are able to eliminate infected macrophages and also to kill intracellular bacteria via production of granulysin (a cytolytic and proinflammatory substance) [28].

Other immune cells—CD1 restricted T cells, γ/δ-T cells, and cytotoxic T-cells—also protect lung tissue against tuberculosis. T cells of type γ/δ are the first line of defense against microbial antigens and the connection between innate and adaptive immune response [29]. They have cytotoxic activity and may present mycobacterial antigens to CD8+ and CD4+ lymphocytes. In response to IL-23 secreted from infected dendritic cells, the γ/δ-Т cells start to release IL-17 for accumulation of additional immune cells in the place of infection.

Activated macrophages represent higher phagocytic activity against extracellular mycobacte‐ ria. On the base of T-lymphocyte stimulation, two types of macrophages are known: classically activated macrophages (САМ) and alternatively activated macrophages (ААM). During the immune response against tubercle bacilli, activation of macrophages by T-lymphocytes is achieved mainly through release of IFN-gamma and other Th-1 cytokines. The Th-1 cytokines (INF-gamma, TNF-alpha, IL-1beta) induce САМ to kill tubercle bacilli via production of nitric oxide synthetase (iNOS). This enzyme catalyzes synthesis of nitric oxide (NO), a powerful antimicrobial substance. The Th-2 cytokines (IL-4 and IL-13) activate ААM to produce antiinflammatory cytokines and arginase. They both compete for arginine utilization with iNOS [30]. The increased arginase activity stimulates tissue repair but at the same time restricts bacterial killing [31].

*M. tuberculosis* (particularly the virulent strains and not the attenuated ones) can survive in phagosomes of macrophages by suppressing the fusion between formed mycobacterial phagosome and lysosomes [32]. Other mechanisms to overcome bactericidal activity of macrophages include the prevention of phagosomal acidification and guidance of infected cells towards necrosis. Necrosis is a form of cellular death characterized by plasma and mitochondrial membrane disruption. In this way, tubercle bacilli leave infected macrophages and spread to other cells. Moreover, *M. tuberculosis* may inhibit the programmed cell death or apoptosis, a cellular death that protects further bacterial spread by preserving the cellular integrity of infected macrophage. Apoptotic macrophages transmit mycobacterial antigens to dendritic cells and induce more efficient T cell response.

#### **3.4. Humoral immunity**

In addition to cell-mediated immunity, humoral immune response plays a major role in TB infection control. The formed antibodies cannot transfer immunity but have an opsoning role and facilitate the phagocytosis by the macrophages and the cytotoxic function of T-lympho‐ cytes [33]. Besides their antibody synthetic function [34], B-lymphocytes can present myco‐ bacterial antigens to T cells [35]. They stimulate proliferation and differentiation of Tlymphocytes by the production of different cytokines. Specific effector B1-cells secrete Th1 cytokines—IFN-gamma and IL-12—while B2-cells secrete IL-4, typical for Th2-cells. Furthermore, activated B cells can produce IL-6 for T cell stimulation and IL-10 for inhibition of dendritic cells and macrophages.

#### **3.5. Latent tuberculosis**

molecules—MHC class I-restricted cytotoxic T-lymphocytes (CTL). They are able to eliminate infected macrophages and also to kill intracellular bacteria via production of granulysin (a

Other immune cells—CD1 restricted T cells, γ/δ-T cells, and cytotoxic T-cells—also protect lung tissue against tuberculosis. T cells of type γ/δ are the first line of defense against microbial antigens and the connection between innate and adaptive immune response [29]. They have cytotoxic activity and may present mycobacterial antigens to CD8+ and CD4+ lymphocytes. In response to IL-23 secreted from infected dendritic cells, the γ/δ-Т cells start to release IL-17

Activated macrophages represent higher phagocytic activity against extracellular mycobacte‐ ria. On the base of T-lymphocyte stimulation, two types of macrophages are known: classically activated macrophages (САМ) and alternatively activated macrophages (ААM). During the immune response against tubercle bacilli, activation of macrophages by T-lymphocytes is achieved mainly through release of IFN-gamma and other Th-1 cytokines. The Th-1 cytokines (INF-gamma, TNF-alpha, IL-1beta) induce САМ to kill tubercle bacilli via production of nitric oxide synthetase (iNOS). This enzyme catalyzes synthesis of nitric oxide (NO), a powerful antimicrobial substance. The Th-2 cytokines (IL-4 and IL-13) activate ААM to produce antiinflammatory cytokines and arginase. They both compete for arginine utilization with iNOS [30]. The increased arginase activity stimulates tissue repair but at the same time restricts

*M. tuberculosis* (particularly the virulent strains and not the attenuated ones) can survive in phagosomes of macrophages by suppressing the fusion between formed mycobacterial phagosome and lysosomes [32]. Other mechanisms to overcome bactericidal activity of macrophages include the prevention of phagosomal acidification and guidance of infected cells towards necrosis. Necrosis is a form of cellular death characterized by plasma and mitochondrial membrane disruption. In this way, tubercle bacilli leave infected macrophages and spread to other cells. Moreover, *M. tuberculosis* may inhibit the programmed cell death or apoptosis, a cellular death that protects further bacterial spread by preserving the cellular integrity of infected macrophage. Apoptotic macrophages transmit mycobacterial antigens to

In addition to cell-mediated immunity, humoral immune response plays a major role in TB infection control. The formed antibodies cannot transfer immunity but have an opsoning role and facilitate the phagocytosis by the macrophages and the cytotoxic function of T-lympho‐ cytes [33]. Besides their antibody synthetic function [34], B-lymphocytes can present myco‐ bacterial antigens to T cells [35]. They stimulate proliferation and differentiation of Tlymphocytes by the production of different cytokines. Specific effector B1-cells secrete Th1 cytokines—IFN-gamma and IL-12—while B2-cells secrete IL-4, typical for Th2-cells. Furthermore, activated B cells can produce IL-6 for T cell stimulation and IL-10 for inhibition

for accumulation of additional immune cells in the place of infection.

dendritic cells and induce more efficient T cell response.

cytolytic and proinflammatory substance) [28].

60 Immunopathology and Immunomodulation

bacterial killing [31].

**3.4. Humoral immunity**

of dendritic cells and macrophages.

The late stage of TB infection represents a prolonged (in many cases lifelong) suppression of mycobacterial cells as a result of the dynamic balance between the pathogen and the host immunity. *M*. *tuberculosis* persists within the granulomas and escapes total disruption by the immune system. In latent TB, the host and tubercle bacilli coexist in "perfect" synergy; the granuloma represents a place for bacterial survival and place for host attack [36]. Usually, the host immune system succeeds to manage the infectious process, but in extreme conditions (starving, diabetes, alcohol abuse, corticosteroid treatment, and especially supplementary HIV infection) the disease can progress. This progression is linked to granuloma disruption and further dissemination of *M*. *tuberculosis*. The late tuberculosis occurs in the presence of sensitized T-lymphocytes and already existing specific defense mechanisms. This determines rapid limitation of the process with strong caseous necrosis, cavern formation, and fibrosis.
