The influenza viruses (types A, B, and C) are negative sense single-stranded RNA viruses (7). These enveloped viruses belong to the family of Orthomyxoviridae. In humans, the most common cause of respiratory illness is influenza A virus (IAV) (8). To reach successful replication, sense messenger RNAs must be generated from the viral genome, a process due to the viral RNA polymerase. Sense messengers comprise eight RNA segments encoding eleven proteins (9, 10). The mature virion contains eight of these proteins surrounded by a protein envelope, which mainly includes two viral antigenic determinants: HA, which binds to terminal sialic acids expressed by airway and alveolar epithelial cells and NA, a critical enzyme necessary for releasing the viral progeny from infected cells. Eighteen HA subtypes and 11 NA subtypes have been identified to date. Two IAV subtypes (H1N1 and H3N2) along with one or two influenza B viruses co-circulate annually causing influenza epidemics. The primary targets (and site of replication) of IAV are airway and alveolar epithelial cells. Shortly after infection, the viral machinery, with the unintentionally help of host factors, is at work and generate the release of virions (11). Productive replication in epithelial cells results in cell death and to epithelial/endothelial damages leading to barrier rupture and exudation of fluids and proteins into the airways and alveolar spaces, greatly impairing gas exchanges (12). Meanwhile, an intense infiltration of immune cells (neutrophils, monocytes) occurs. Clinically, severe IAV infection can lead to acute respiratory distress syndrome, a severe form of respiratory failure associated with 40 % of mortality (5).

Natural killer T (NKT) cells represent a subset of lipid-reactive αβ T cells. In response to lipid Ags presented by the monomorphic Ag presenting molecule CD1d, NKT cells swiftly produce a large amount of cytokines, thus promoting and orientating immune responses (77). Lipid recognition by NKT cells is mediated by a conserved T cell receptor (TCR) repertoire. Natural killer T cells can be divided into two major populations: type I NKT cells and type II NKT cells. Type I NKT cells express a semi-invariant TCR α-chain (Vα14-Jα18 in mice and Vα24-Jα18 in humans) paired with a limited set of TCR β-chains (77, 78). These cells respond strongly to alpha-galactosylceramide (α-GalCer), a glycolipid under clinical development, particularly in cancer settings (79). Type I NKT cells also recognize endogenous lipids which are necessary for their selection in the thymus and for their activation at peripheral sites. Type I NKT cells can also react to microbial-derived lipids (80). Of importance, type I NKT cells also activate in response to a wide array of cytokines, including IL-12 and IL-23. Despite a relatively conserved TCR, type I NKT cells are heterogeneous and can be further divided into distinct subsets (81, 82). NKT cells produce a wide range of cytokines, with sometime opposite functions, a property that depends on the cell subset activated and on the nature of the stimulation (e.g., lipids and/or activating cytokines). Through this unique property, type I NKT cells can influence different types of immune responses ranging from T helper (Th)1-like, Th2-like, Th17-like, or T regulatory-like responses (83). This property is critical in pathological situations during which type I NKT cells can either exert positive or negative functions. Of note, type I NKT cells not only produce cytokines and display cytotoxic functions toward transformed cells and virally-infected cells (84). Type II NKT cells represent a much broader family of CD1d-restricted αβ T cells that react to lipids, but not to α-GalCer. They express a more diverse TCR repertoire that recognizes lipid Ags of various nature and origin (mammalian and microbial) (85). Due to the lack of specific tools, the functions of type II NKT cells have mainly been proposed indirectly by comparing the phenotypes observed in Jα18-deficient (which lack type I NKT cells) vs. CD1d-deficient (which lack both type I and type II NKT cells) mice in various settings. Type II NKT cells appear to share conserved phenotypic and functional features with type I NKT cells including an effector memory phenotype, cytotoxic potential and secretion of numerous cytokines/chemokines (85). Akin to type I NKT cells, type II NKT cells play important functions during (bacterial) infections. NKT cells, which are more abundant in mice relative to humans, populate both lymphoid tissues and mucosal sites, including the lungs (86, 87).

Mucosal-associated invariant T (MAIT) cells present many common features with NKT cells and γδ T cells including the capacity to rapidly react to non-peptide Ags. MAIT cells are defined by their restriction to the major histocompatibility complex class I-related molecule 1 (MR1) (88, 89). The majority of MAIT cells (referred to as classical MAIT cells) (90) express a semi-invariant TCR composed of a canonical TCRα-chain (Vα19-Jα33 in mice and Vα7.2-Jα33 in humans) associated with a restricted set of Vβ segments (88, 89, 91, 92). Through their TCR, MAIT cells recognize small intermediate metabolites from the riboflavin (vitamin B2) pathway of bacteria, mycobacteria and yeast (93–95). They can react to products derived from the non-enzymatic reaction between a riboflavin precursor and small aldehydes of both microbial and host origin. The high instability of these ligands has so far limited their use in the clinics. Reminiscent with NKT cells, MAIT cells can respond to TCR signals and/or to various activating cytokines, including IL-12 and IL-18 (96–98). Upon activation, MAIT cells produce large amounts of Th1- and Th17-related cytokines (99). Additionally, MAIT cells can kill bacteria-infected cells (100). Unlike NKT cells, MAIT cells are abundant in the blood (up to 10% of the T cell compartment) in humans. They are also present at mucosal sites, including the lungs (10% of respiratory mucosal T cells) (101), where they sense the environment and exert a role of sentinels of the immune system. Due to their scarce representation in common laboratory mouse strains (unlike NKT cells), understanding MAIT cell biology is challenging even using Mr1^−/− mice. To better assess their role in preclinical models, transgenic mice (Vα19iTg x Cα^−/−) displaying high content of MAIT cells have been developed (102). Given their cytokine profile and cytotoxic potential, MAIT cells intuitively emerged as a specialized cell population in host defense against bacteria.

γδ T cells represent approximately 1–10% of peripheral blood T cells in humans and are important components of both innate and adaptive immunity. They display vast effector and immune regulatory functions (103). Akin to other members of the unconventional T cell family, γδ T cells display a pre-activated status that allows rapid induction of effector functions following the detection of tissue stress (104–106). Another important feature of γδ T cells is their tropism for epithelial surfaces including lungs, to where they migrate shortly after development and persist as resident cells. They frequently express invariant or closely related γδ TCRs in a given tissue site (e.g., Vγ1, Vγ4, and Vγ6 in the mouse lung tissue), which confer them specific Ag recognition capabilities from one tissue to another (103). Reminiscent to other unconventional T cells, γδ T cells can kill infected cells and initiate adaptive immune responses through the release of substantial amounts of Th1- and Th17-related cytokines (103). Thus, γδ T cells have emerged as essential constituents of the antimicrobial immunity in both preclinical and clinical settings (107). While the Ags for mouse γδ TCRs have not been reported yet, human γδ T cell subsets (e.g., Vγ9Vδ2⁺ cells) can recognize both natural (of microbial and mammalian origins) and synthetic phosphoantigens (108). However, it is now clear that the phosphoantigens are not directly sensed by the γδ TCRs but rather require the involvement of butyrophilin BTN3A as an intermediate. The precise molecular mechanisms involved in this TCR-dependent γδ T cell activation are still a matter of debate (109). Whatever the mechanisms involved, phosphoantigens have been shown to strongly activate (in vivo and ex vivo) human Vγ9Vδ2 γδT cells to induce their proliferation and to increase their cytotoxic capacities as well as their cytokine secretion including IFN-γ and TNF-α. Given this, harnessing γδ T cell functions in therapeutic protocols is currently highly considered by clinicians especially in the context of cancer (110).

Streptococcus pneumoniae (also referred to as the pneumococcus) is the leading cause of community-acquired bacterial pneumonia worldwide (2 million deaths per year), with infants and the elderly exhibiting higher susceptibility. This Gram-positive bacterium, which comprises a group of more than 90 serotypes, colonizes asymptomatically nasopharynx of healthy individuals. However, when the immune equilibrium is broken, pneumococcus carriage can lead to mild disease such as otitis media or sinusitis and more occasionally turns into severe complications such as pneumonia, sepsis, and meningitis (111). Streptococcus pneumoniae is often found in biological fluids of hospitalized patients diagnosed for influenza infection as well as patients with exacerbated chronic lung inflammation (112, 113). Despite vaccination prevents pneumococcus spread and controls infections, the available vaccines have however some issues (114). In addition, the emergence of antibiotic-resistant strains represents an important threat for the management of pneumococcal infections in clinics (115).

In the mouse system, both type I NKT cells and γδT cells activate in response to S. pneumoniae (Figure 1). While type I NKT cells produce IFN-γ early after pneumococcal challenge, γδT cells produce IL-17A (66, 74, 116–118). Activation of type I NKT cells during S. pneumoniae infection depends on pneumococcal-derived lipid(s) (α-glucosyldiacylglycerol), cytokines (IL-12) or both according to the strain studied (86, 116, 119). Of note, we and others have highlighted the role of CD103⁺ dendritic cells in the activation of type I NKT cells during pneumococcal infection (119, 120). The mechanisms through which murine γδT cells activate (IL-17) mainly depends on IL-1β and IL-23 (117, 121). The lack of type I NKT cells (Jα18^−/− mice) (119, 122, 123) or γδT cells (Tcrd^−/− mice) (66, 117) results in higher bacterial loads and mortality. The underlying mechanisms of this protective activity rely on IFN-γ and IL-17 secretion and on the early recruitment of neutrophils. Hence, both type I NKT cells and γδT cells play a natural positive role in host defense against experimental pneumococcal infection (Figure 1). The potential role of type II NKT cells and MAIT cells during pneumococcal infection is still elusive. Of interest, S. pneumoniae expresses enzymes involved in the synthesis of riboflavin metabolites (124, 125) and human MAIT cells produce, in an MR1-dependent manner, IFN-γ in response to dendritic cells and airway epithelial cells exposed to S. pneumoniae (126, 127). Using Vα19iTg x Cα^−/− mice, a small proportion of lung MAIT cells were shown to produce IFN-γ and IL-17A during pneumococcal infection (127). Although a more detailed kinetic analysis is required, these levels were relatively low compared to those produced by NKT cells and γδT cells. The use of Mr1^−/− or Vα19iTg x Cα^−/−Mr1^−/− mice will be instrumental to address the role of MAIT cells during experimental pneumococcal infection.

The potential effects of exogenous activation of unconventional T lymphocytes on pneumococcal infection have been examined. Inoculation of the type I NKT cell superagonist α-GalCer protects against lethal pneumococcal infection in the mouse system (123, 128). Mechanistically, this protective activity relies on respiratory CD103⁺ dendritic cells and on both IFN-γ and IL-17A production and neutrophils (128). The potential effect of exogenous γδ T cell and MAIT cell activation on host defense against pneumococcal infection is presently unknown. Despite emerging evidence for a critical role in host response to pneumococcus in experimental models, information regarding the phenotype and dynamics of unconventional T cells in patients with severe S. pneumoniae-driven pneumonia are rather limited. Of note, the level of circulating MAIT cells in critically ill patients with severe bacterial infection is markedly decreased compared to age-matched healthy controls (129). Although this decrease is less striking in patients with streptococcal infections, these data suggest that MAIT cells may migrate into the lungs, and thus may exert a potential role during pneumococcal infection.

Staphylococcus aureus is a Gram-positive bacterium with a potent pathogenic potential to cause a variety of community and hospital-acquired infections. In normal conditions, it commonly colonizes the upper airways. Under certain circumstances, including influenza infection, it can cause localized and serious invasive infections, as well as a severe septic shock syndrome (130). The frequency of these infections is increasing. The ability of S. aureus to form biofilms and the emergence of multidrug-resistant strains (e.g., methicillin-resistant Staphylococcus aureus) are the main reasons why their treatment is becoming more difficult. The capacity of S. aureus to become pathogenic is related to the expression of virulence factors, among which the production of a wide variety of toxins. Staphylococcal superantigens (SAgs) constitute a family of potent exotoxins secreted by S. aureus (131). They can cross-link MHC class II molecules with TCRs to stimulate an uncontrolled polyclonal activation of T lymphocytes (cytokine storm), potentially leading to severe illnesses including toxic shock syndrome.

Despite a relatively poor literature in the field, unconventional T cells might play role during S. aureus infection. They also recently emerged as potential targets of Staphylococcal SAgs. Indirect evidence suggest that IL-17 production by γδ T cells might be important in the control of S. aureus lung infection (72, 73) (Figure 1). Mice lacking γδ T cells have a reduced ability to clear bacteria and to control pulmonary inflammation (132). The role of NKT cells and MAIT cells in the control of S. aureus is still unknown. On the other hand, emerging evidence suggest that unconventional T cells (at least NKT cells and MAIT cells) are involved in toxic shock syndrome induced by Staphylococcal SAgs. Intranasal inoculation of Staphylococcal enterotoxin B promotes the activation of type I NKT cells and lung injury (133). Staphylococcal enterotoxin B activates mouse and human type I NKT cells via a MHC class II (but not CD1d) Vβ8-dependent pathway (134). More recently, Szabo et al., using SAg-sensitive HLA-DR4-transgenic mouse demonstrated that type I NKT cells are pathogenic (toxic shock syndrome) in response to Staphylococcal enterotoxin B (135). Of interest, administration of a Th2-polarizing glycolipid agonist for type I NKT cells reduced morbidity and mortality. Type I NKT cells may therefore constitute an attractive therapeutic target in SAg-mediated illnesses. Mouse and human MAIT cells can also activate in response to Sags in a largely TCR-independent, cytokine-driven manner (136). They produce a huge amount of pro-inflammatory cytokines and thereafter become unresponsive to stimulation with bacterial Ags. Through this mechanism, they might participate in cytokine storm and subsequent immunosuppression. Akin to type I NKT cells, MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses.

Disruption of the pulmonary barrier functions strongly contributes to enhanced bacterial colonization, bacterial superinfection and bacterial pneumonia in the context of prior influenza. Emerging evidences suggest that unconventional T cells play a natural role in the maintenance of tissue integrity and/or in tissue repair processes (138). Recent studies have addressed the role of unconventional T cells in tissue homeostasis and barrier functions during experimental influenza. Type I NKT cells and γδ T cells produce the tissue protective cytokine IL-22 (through IL-1β- and IL-23) during the early course of IAV infection (24, 139). Although IL-22 does not affect viral loads during influenza, several independent groups have demonstrated the protective effect of IL-22 against epithelial damages caused by viral replication (24, 139–143). The mechanisms through which IL-22 prevents epithelial barrier dysfunction during influenza infection might include an inhibitory effect on the recruitment of inflammatory monocytes and a direct effect on the expression of genes involved in barrier functions (143). Interleukin-22 might also participate in airway epithelial regeneration and barrier repair (141, 142). Interestingly, through its protective effect on barrier functions, IL-22 reduces secondary bacterial infection (139, 143). Of note, MAIT cells have recently been reported to accumulate in the lungs and to activate (through IL-18) during experimental IAV infection, a process associated with protection against a lethal viral challenge (33). Although not firmly established, MAIT cell activation during IAV infection may reduce pulmonary epithelial damage and reinforce barrier functions. Hence, by rapidly producing tissue protective factors, unconventional T cells, including NKT cells, γδ T cells, and MAIT cells, may limit the extent of secondary bacterial infection post-influenza. It is noteworthy that NKT cells can also indirectly activate the synthesis of protective barrier factors by other cells (e.g., amphiregulin by group 2 innate lymphoid cells) (25, 41). These functions might be exploited fortherapeutic purposes.

Alteration of innate immune defense also strongly contributes to bacterial superinfection post-influenza. As stated above, unconventional T cells play a part in host defense against S. pneumoniae and S. aureus. Here, we summarize host factors that (may) compromise their protective functions in the context of double viral and bacterial infection (mouse system). In this setting, IL-10 and type I IFNs appear to play a relevant role. During IAV infection, IL-10 is massively produced by innate and adaptive immune cells. This includes CD4⁺ (including regulatory T cells) and CD8⁺ T cells as well as NK cells and myeloid cells, mostly inflammatory monocytes (38, 119). Our data indicate that in the context of prior influenza, type I NKT cells fail to produce the protective cytokine IFN-γ (Figure 2), an effect associated with worse secondary pneumococcal infection (119). Blockade of IL-10 rescues activation of type I NKT cells (through restoration of IL-12 production by Ag-presenting cells), reduces bacterial outgrowth and dissemination and improves disease outcomes. Hence, the lack of type I NKT cell activation participates, at least in part, to bacterial (pneumococcal) superinfection post-influenza. Along with IL-10, type I IFNs favor bacterial superinfection post-influenza (57, 66, 68, 72–74, 144). γδ T cells appear to be the main target of type I IFNs. In the context of double IAV-bacterial (both pneumococcal and staphylococcal) infection, γδ T cells fail to secrete IL-17 in a type I IFN-dependent manner (66, 72, 74) (Figure 2). This ultimately leads to altered neutrophil recruitment and activity and to inhibition of the IL-17 antimicrobial pathway, including production of antimicrobial peptides. In this setting, the mode of action of type I IFNs is multiple. Type I IFNs can block the secretion of Th17-promoting cytokines IL-1β and IL-23 by Ag-presenting cells (72, 73). On the other hand, type I IFNs can directly target γδ T cells, via IFNAR, to inhibit IL-17 production (74) (Figure 2). Finally, type I IFNs indirectly inhibit IL-17 release by γδ T cells by promoting IL-27 production (66, 67). IL-27 targets γδ T cells to decrease expression of the IL-17-promoting factors RORγt and IL-23 receptor (66). The later mechanism is probably dominant as exogenous administration of IL-27 reverses the resistance phenotype of IFNAR-deficient mice upon post-influenza bacterial infection via down-regulating IL-17 production by γδ T cells and neutrophil response. Whether MAIT cell functions are affected by influenza, for instance through IL-10 or type I IFNs, is still ignored. Type III IFNs have also been shown to favor bacterial superinfection post-influenza (70). Regarding the role of unconventional T cells in barrier functions and innate antibacterial immunity, one can speculate that type III IFNs (like type I IFNs) also alter the functions of these cells to favor bacterial superinfection.

Whilst the use of experimental models suggests a role for NKT cells and γδ T cells in bacterial superinfection post-influenza (the potential role of MAIT cells has not yet been appreciated), few studies have so far investigated unconventional T cells during human influenza and secondary infections. MAIT cells are more abundant in the blood relative to NKT cells and, to a lesser extent, γδ T cells. Compared to healthy donors, the frequency of circulating MAIT cells decreased in patients hospitalized for severe pneumonia due to infection with the Asian lineage avian IAV (H7N9) (96). Of interest, individuals who recovered from pneumonia had a higher level of circulating MAIT cells compared with patients who succumbed (96). This study suggested a protective role of MAIT cells in human influenza. Another clinical study confirmed the reduced peripheral blood MAIT cell frequencies (and enhanced granzyme B expression) in patients with acute IAV infection (2009 H1N1 pandemic) (145). This decrease was even more pronounced in critically ill patients admitted in intensive care unit compared to patients with mild symptoms. Reduction of MAIT cell numbers during acute human influenza infection (critically ill patients) could impair protective anti-bacterial immunity increasing the risk of secondary bacterial infections, which would enhance disease severity and mortality. The frequency, number and functional state of NKT cells and γδ T cells during human influenza have not yet been examined. In influenza vaccinated individuals, γδ T cells proliferate and activate although this intensity weakens with age (146).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.