Cytokines induce effector T-helper cells during invasive aspergillosis; what we have learned about T-helper cells?

Invasive aspergillosis caused by Aspergillus species (Aspergillus fumigatus, A. flavus, and A. terreus) is life-threatening infections in immunocompromised patients. Understanding the innate and adaptive immune response particularly T-helper cells (TH-cells) against these Aspergillus species and how the different sub-set of TH-cells are regulated by differentiating cytokines at primary target organ site like lung, kidney and brain is of great significance to human health. This review focuses on presentation of Aspergillus through Antigen presenting cells (APCs) to the naive CD4+ T-cells in the host. The production of differentiating/effector cytokines that activate following TH-cells, e.g., TH1, TH2, TH9, and TH17 has been reported in association or alone in allergic or invasive aspergillosis. Chemokines (CXCL1, CXCL2, CCL1, and CCL20) and their receptors associated to these TH-cells have also been observed in invasive aspergillosis. Thus, further study of these TH-cells in invasive aspergillosis and other elements of adaptive immune response with Aspergillus species are required in order to have a better understanding of host response for safer and effective therapeutic outcome.


Introduction
Fungi are the most common microorganisms and have clinical importance. Few of them are pathogenic or opportunistic pathogen and results in morbidity and mortality to human beings. There is a rise in opportunistic fungal infections in recent years due to increased incidence of immunocompromised host (Chamilos et al., 2006;Romani, 2008). After Candida albicans, the leading causes of fungal infections in immunocompromised individuals are from Aspergillus species. Aspergillus is one of the most ubiquitous medically important opportunistic fungi (Weaver et al., 2007). The genus Aspergillus, contains about 40 species that can cause infection (Verweij and Brandt, 2007), among them A. fumigatus, A. flavus, and A. terreus are the leading cause of invasive Aspergillosis in immunocompromised individuals. These species produce conidia at a concentration of around 1-100 conidia per m 3 (Barnes and Marr, 2006). Human routinely inhale hundreds of these conidia per day, despite these exposure to Aspergillus conidia, human do not develop any disease due to the clearance of conidia from lung by innate immunity especially phagocytic cells (Chamilos et al., 2006;Romani, 2008). However, due to rise in immunocompromised host, e.g., patients receiving organ transplant, immunosuppressive therapy for autoimmune or neoplastic disease and HIV patients, inhaled conidia if not cleared in these host, colonization of Aspergillus occurs (Stevens et al., 2000). The adaptive immune response in human responsible for conidia clearance is not well understood in immunocompetent host as well as where conidia colonize in immunocompromised host. It is worth to note that secondary metabolites (e.g., Gliotoxins, Aflatoxins) excreted by Aspergillus especially have been recognized to modulate immunological responses (Shankar, 2013). Thus, we reviewed recent advances made in immune responses against Aspergillus species in mice model studies and clinical aspergillosis patients. A. fumigatus is the prominent species, which cause 90% of Aspergillosis followed by A. flavus and A. terreus. Studies have showed that Aspergillus species associated with infection after hematopoietic stem cell transplantation include A. fumigatus with 56% followed by A. flavus (18.7%) and A. terreus (16%) (Steinbach et al., 2004;Morgan et al., 2005). The involvement of Aspergillus infection in pulmonary tuberculosis and in asthmatic patients has been reported by Denning et al. It has been estimated annually at least 372,385 patients developed chronic pulmonary aspergillosis worldwide following treated pulmonary tuberculosis (Denning et al., 2011). Similarly, around 4,837,000 patients develop Allergic bronchopulmonary aspergillosis out of 193 million adults with active asthma (Denning et al., 2013). However, in a recent study of Indian population by Agarwal et al. (2014) the estimated ABPA burden was 1.38 million out of 27.6 million adults with asthma. This review touches different aspect of antifungal immunity against aspergillosis that include antigen presenting cells (APCs), dendritic cells (DCs), fungal pattern recognition receptors (PRRs), T H -cells with their subsets profile during infection associated to Aspergillus species at different site of infection, e.g., lung, kidney, and brain.

Recognition of Aspergillus by the Host Presentation of Pathogen via Soluble Receptors and Cell Bound Receptors
After the inhalation of A. fumigatus conidia, they are entrapped by the lung alveoli and if they are not efficiently cleared from lung, they germinate and establish lung infection termed invasive pulmonary aspergillosis and it may also disseminates to other organs if not treated ). The recognition of A. fumigatus conidia and hyphae occurs by PRRs those include soluble receptors and cell-bound receptors. Conidial germination starts with hydrophobic layer degradation and exposure of inner cell wall components mainly polysaccharides, which includes chitin, β-glucan, mannan, and galactomannan. These are termed as pathogen associated molecular patterns (PAMP), are recognized by PRRs (Netea et al., 2006;Inoue and Shinohara, 2014). PRRs soluble receptor such as pulmonary collectins, family of C-type lectins, pentraxin-3, pulmonary surfactant proteins-A and D have been reported in aspergillosis. Further, the cellbound receptors in association with aspergillosis include Toll like receptor-2 (TLR), TLR-4 and TLR-9, which potentially induce the production of pro-inflammatory cytokines and reactive oxygen species through MyD88 signaling pathway (Willment and Brown, 2008).

Antigen Presenting Cells and T-cell Differentiation Antigen Presenting Cells Triggers Cytokines Production
The activation of the innate immunity through PRRs present on the APCs that regulate the development of T-cell. APCs express wide-array of PRRs that provides the link between adaptive and innate immunity ). APCs, dominantly DCs, are responsible for antigen monitoring and then shaping Tcell response by secreting cytokines and chemokines. DCs express PRRs on their cell surface and endosomal compartments, which serve to recognize PAMPs. After interaction with DCs, naive Tcells are activated. The activation of T-cell response is regulated by the cytokines milieu predominantly framed by DCs (Akdis et al., 2011). Chemokines secreted by DCs recruit the phagocytic cells to infected areas to clear the Aspergillus components. APC cells, e.g., monocytes differentiate into distinct sub-populations of CD14 + and CD16 + cells after A. fumigatus conidia infection (Serbina et al., 2009). Monocytes interact with Aspergillus antigens resulting in maturation of monocytes into macrophages or DCs. Macrophages and DCs interact with antigens and secrete effector cytokines (Osugi et al., 2002;Ramirez-Ortiz and Means, 2012). Major sub-populations of DCs are myeloid DCs, plasmacytoid DC (pDCs) and monocyte-derived DCs (Bozza et al., 2002;Osugi et al., 2002). pDC recognize the nucleic acids from A. fumigatus via TLR-9 and lead to resistance to A. fumigatus infection in mice (Ramirez-Ortiz et al., 2008). Further, monocytes migrate toward the lung to differentiate into either DCs or alveolar macrophages during invasive aspergillosis (Cramer et al., 2011;Morton et al., 2012). Monocytes express different chemokines receptor predominantly CCR2, which help in migration of monocytes from bone marrow toward lung in response to A. fumigatus infection (Serbina et al., 2009). It has been shown that monocytes expressing CCR2 in the lung involved in conidial uptake and killing (Espinosa et al., 2014). Furthermore, alveolar macrophages induce APCs to release IL -1β in pulmonary invasive infection ). IL-18 has also been observed in lung during invasive aspergillosis mice model (Akdis et al., 2011). Recently, it has been observed that A. fumigatus pulmonary challenge induces expression of the inflammasome-dependent cytokines IL-1β and IL-18 within the first 12 h, while IL-1α expression continually increases over at least the first 48 h (Caffrey et al., 2015). Moretti et al. (2014) showed in a pulmonary invasive aspergillosis model that mice injected with IL-37 prior to A. fumigatus infection has significant reduction in IL-β production and recruitment of neutrophils and resulted in diminution in lung inflammation and damage. The anti-inflammatory activity of IL-37 has been observed as an inhibitor of the innate response. Thus, cytokines play a vital role in modulation of immune response and coordinate the innate as well as adaptive responses. APCs secrete cytokines that act on naïve T-cells leading to the differentiation of naïve Tcells. These differentiated T-cells further secrete effector cytokines and regulate the function of T H -cells. The profile of cytokine depends on the type of Aspergillus antigens, route of infection, immunological status of the host and cytokines milieu present during the interaction (Romani, 2008;Chai et al., 2010b). CD4 + T-cells can be divided into distinct subtypes according to cytokine profile, and they can differentiate to T H 1, T H 2 T H 17, T H 9, and Tfollicular effector cells (Kerzerho et al., 2013;Kara et al., 2014). On the basis of the cytokine profile, these T H -cells perform distinct functions. However, it is not clear how T-follicular effector cells respond during Aspergillus infection (Wüthrich et al., 2012).

Cytokines Associated with T H 1 Type of Response
Aspergillus fumigatus challenged intranasally in mice interacts with DCs and alveolar macrophages in the lung. Secretion of T H 1 associated pro-inflammatory cytokines IL-12, IFN-γ, TNFα, IL-18 has been observed after the challenge (Chotirmall et al., 2013). Among these, IL-12 is the prominent cytokine released from activated monocytes and macrophages in lung that help in shaping T-cell immune response. IL-12 is a heterodimeric cytokine composed of IL-12p35 and IL-12p40 polypeptides that form the bioactive IL-12p70. The heterodimer binds to the IL-12 receptor composed of IL-12Rβ1 and IL-12Rβ2 chains and signals through STAT-4 (Shao et al., 2005). IL-12 acts on NK cells to promote IFN-γ secretion and differentiate CD4 + T-cells into T H 1-cells, once CD4 + cells differentiates to T H 1-cells, they increase the secretion of IFN-γ, which suppress T H 17 and T H 2 response in the lung (Espinosa and Rivera, 2012;Camargo and Husain, 2014). IL-12, hence, is the most important regulator of T H 1 response during lung infection. IL-12 deficient mice failed to generate a T H 1 response, leading to increased secretion of IL-4 and IL-10 cytokines, which shifts the immune response toward T H 2 pathway (Cenci et al., 1998). In A. fumigatus induced neutropenic aspergillosis in mice, NK cells can be the primary source of IFN-γ responsible for activating phagocytic cells and direct antifungal effectors cells against A. fumigatus . Further, patients with invasive Candida and/or Aspergillus infections, recombinant treatment of IFN-γ in combination with antifungal drug partially restored immune function (Delsing et al., 2014). In intravenous infection of A. flavus mice model studies, lung and brain homogenate showed pro-inflammatory cytokines IL-12 and IFN-γ and relative absence of IL-4, IL-23, and IL-17 suggesting a T H 1 response (Anand et al., 2013(Anand et al., , 2015. A. terreus induced invasive aspergillosis showed the presence of IL-1β, IL-6, and reduced level of IL-10 in mice model studies. Although there is activation of T H 17 type of adaptive immune response through IL-1β but the later is suppressed by T H 1 cytokines particularly IFN-γ (Vyas, 2011;Lass-Florl, 2012). The T H 1 response is thus also mounted by A. terreus and there is a lack of T H 2 response in contrast to A. fumigatus infection where T H 2 promoting cytokines are observed. (Chai et al., 2010b). Activation of T H 17-cell depends on Dectin-1 signaling pathway. Various studies have suggested that dectin-1 deficient mice entirely activate T H 1cells. So Dectin-1 signaling not only serves as a positive factor to promote T H 17 differentiation but rather act to balance T H 1 versus T H 17 differentiation. Activation of the APCs by Dectin-1, release the proinflammatory cytokines IL-1β, IL-6, IL-23, and IL-22 which differentiates CD4 + T-cells to T H 17-cells, which further secretes IL-17A and IL-17F cytokines and maintain T H 17 response (Werner et al., 2011). IL-23 is a member of IL-12 family, produced by phagocytic cells, macrophages and activated DCs in lung. IL-23 contains two subunits IL-12p40 and IL-23p19 and it binds to heterodimeric receptors IL-12Rβ1, expressed by activated T-cells (Zelante et al., 2007). IL-6 is another important cytokine involved in regulation of T H 17 response. IL-6 is a multifunctional cytokine, promote T H 17-cells differentiation, inflammation and acute response (Akdis et al., 2011). During T H 17 differentiation, human naïve T-cells are exposed to IL-1β, IL-6, and IL-23 (Zelante et al., 2009;Gresnigt and van de Veerdonk, 2014). T H 17 promoting cytokine IL-17 binds to IL-17RA and IL-17RC receptors expressed in lung cells, like fibroblast, epithelial cells and T-cells. After release of IL-17 from T H 17-cells, it activates the neutrophils migration toward infected area and increases inflammation (Wilson et al., 2007). In A. flavus and A. terreus, the role of T H 17-cells during lung infection is yet to be established.

Cytokines Associated with T H 2 Type of Response
Aspergillus fumigatus is associated with both invasive and allergic form of aspergillosis. In case, if conidia are not cleared, they germinate to produce hyphae, which are responsible for invasion in host tissues that leads to inflammation. In a healthy human T-cells response, A. fumigatus not only evoke pro-inflammatory type of immune response via T H 1 and T H 17-cells but also antiinflammatory type of immune response mediated by T H 2-cells (Chaudhary et al., 2010). Immune response initiated by IL-4 and IL-10 inhibits T H 1 and T H 17 response and increased secretion of IL-4 and IL-10 inhibits IFN-γ and IL-12 production. T H 2-cells differentiation depends on IL-4 and IL-10 and after differentiation in to T H 2 cells, these cells further secretes IL-5 and IL-13 which maintain T H 2 response. T H 2 immune response is triggered in acute bronchopulmonary aspergillosis and also in invasive pulmonary infection during some time point of infection. IL-4 and IL-10 deficient mice show lower A. fumigatus burden and increased survival rates compared to wild type mouse in invasive pulmonary aspergillosis (Cenci et al., 2000). It has been shown that ESTs (L3 ribosomal protein, L7A ribosomal protein, Histone -H2A) have high sequence similarity with human counter parts suggesting molecular mimicry between human and pathogen protein (Shankar et al., 2004). However, role of these genes in eliciting allergic immune response needs investigations. In A. flavus mediated infection, lung homogenate showed the absence of T H 2 response in a limited cytokine profile study (Anand and Tiwary, 2010). However, T H 2 type response may get activated in later stages of infection in lung due to rise in IL-4 and IL-10, which suppress the T H 1 response but consistent expression IFN-γ overcomes T H 2 response. In addition to T H 2, the role of T H 9-cells has been shown during infection with a Virus, bacteria, parasites and fungi. T H 9 response contributes to allergic inflammation during allergic aspergillosis due to A. fumigatus in mice model (Kerzerho et al., 2013). T H 9-cells develop in the presence of IL-1α and TGF-β along with T H 2-cells. However, role of T H 9 and T H 2 response during invasive aspergillosis remains unclear.

Is Their Co-evolution of T-helper Cells During Invasive Aspergillosis?
In a immunocompromised mice model studies, repeated exposure of A. fumigatus conidia in hosts lead to co-evolution of T H 1, T H 2, and T H 17 response in infected lung (Murdock et al., 2011). They have observed the presence of IFN-γ and IL-17 in infected lung of mice along with T H 2 response. These mixed responses might be occurring at different time points during progression of infection and leads to either protection or infection. T H 1 and T H 17 response probably leads to protection where as T H 2 response further complicates the disease. T H 2 response help in evasion of A. fumigatus from immune cells and further increase the IgE level, which leads to high inflammation at the site of infection. Mice model of ABPA demonstrated the T H 2 cytokine profile consisting of IL-4, IL-10, and IL-5 (Latge, 1999).

Interplay of Cytokines; T H 1 or T H 2 or T H 17 Type of Response
T H -cells response during invasive Aspergillus depends on differentiating cytokines. T H 1 response is activated by differentiating cytokine IL-12 followed by secretion of IFN-γ. Secretion of IFN-γ further stimulates T H 1 cells, if IFN-γ dominates initially it suppress the other cytokines of T H 2 and T H 17, i.e., IL-4 and IL-17 (Harrington et al., 2005). If IL-4 dominates during initial period of Aspergillus infection, it suppresses the protective T H 1 type immune response by inhibiting differential cytokine IL-12 and IFN-γ (Harrington et al., 2005). Recognition of Aspergillus antigens by Dectin-1 signaling pathway inhibit the production of IFNγ and IL-12 receptors suppressing T H 1, which leads to differentiation of T H 17-cells and production of IL-17. In this way Dectin-1 signal balances the T H 1 and T H 17 response through the regulation of their respective cytokines ; Figure 1). The development of effective CD4 + T H -cells response not only depends upon cytokines, but also on chemokines and their receptors. Chemokines help in recruitment of leukocytes, i.e., neutrophils, monocytes and NK cells toward lung during Aspergillus infection. These cells express chemokine receptors; neutrophils contain CXCR2 chemokine receptor for ligand CXCL1 and CXCL2, monocytes contain CCR2 and CCR6 receptor for CCL2 and CCL20 ligands, where as NK cells contain CCR2 receptor for CCL2 ligand. So these chemokine ligands attract monocytes, neutrophils and NK cells to clear the Aspergillus hyphae during lung infection ). These chemokines receptors are also present on DCs, regulatory T-cells (Tregs) and T Hcells and help in their trafficking (Bendall, 2005). In this way, there is an interdependent relationship between chemokines and cytokines that help in evolution of effector T H -cells response. CCL17, a chemokine, help in trafficking of DCs, Tregs and T H 1cells toward infected area during invasive aspergillosis in response to CCR4 chemokine receptor present on these cells. Further, CCR6 receptor present on DCs and T H 17-cells help in migration of these cells in response to chemokine CCL20 (Bendall, 2005;Wüthrich et al., 2012).

Conclusion
Cytokines are important in the development of CD4 + T H -cells. Understanding of trafficking of CD4 + T H -cells and their regulation through differentiating/effector cytokines during invasive aspergillosis will be crucial for the targeted immunotherapy. Overall, cytokines and chemokines may serve as prognostic biomarkers that could be followed to assess the effectiveness of treatment response during invasive aspergillosis. Measurement of selected cytokines in the blood samples of aspergillosis patients may be a promising tool for the monitoring of treatment responses. Also, manipulation of cytokine response e. g, IFN-γ or IFN-γ in combination with antifungal drug, IL-37, may be a future avenue for the development of better therapeutic against invasive aspergillosis.