FEMS Pathogens and Disease, 73, 2015, ftv081 doi: 10.1093/femspd/ftv081 Advance Access Publication Date: 2 October 2015 Minireview

MINIREVIEW

Review of the neutrophil response to Bordetella pertussis infection Joshua C. Eby∗ , Casandra L. Hoffman, Laura A. Gonyar and Erik L. Hewlett Division of Infectious Diseases, University of Virginia, Charlottesville, VA 22908, USA ∗ Corresponding author: Division of Infectious Diseases, University of Virginia, PO Box 800419, Charlottesville, VA 22908, USA. Tel: +(434)924-9633;

Fax: +(434)924-0075; E-mail: [email protected] One sentence summary: Bordetella pertussis suppresses the neutrophil response early after inoculation, but bacterial and host factors combine to promote late neutrophil recruitment, a response that is important for clearance of the bacterium. Editor: Nicholas Carbonetti

ABSTRACT The nature and timing of the neutrophil response to infection with Bordetella pertussis is influenced by multiple virulence factors expressed by the bacterium. After inoculation of the host airway, the recruitment of neutrophils signaled by B. pertussis lipooligosaccharide (LOS) is suppressed by pertussis toxin (PTX). Over the next week, the combined activities of PTX, LOS and adenylate cyclase toxin (ACT) result in production of cytokines that generate an IL-17 response, promoting neutrophil recruitment which peaks at 10–14 days after inoculation in mice. Arriving at the site of infection, neutrophils encounter the powerful local inhibitory activity of ACT, in conjunction with filamentous hemagglutinin. With the help of antibodies, neutrophils contribute to clearance of B. pertussis, but only after 28–35 days in a na¨ıve mouse. Studies of the lasting, antigen-specific IL-17 response to infection in mice and baboons has led to progress in vaccine development and understanding of pathogenesis. Questions remain about the mediators that coordinate neutrophil recruitment and the mechanisms by which neutrophils overcome B. pertussis virulence factors. Keywords: Bordetella pertussis; neutrophil; vaccine; IL-17; toxin

INTRODUCTION Bordetella pertussis is the causative agent of whooping cough, or pertussis, a vaccine-preventable human disease characterized by coughing. Despite high levels of vaccination, the incidence of pertussis in the United States has increased over the last 30 years, raising questions about the mechanisms of disease and basis of immunity (Cherry 2013). Studies of pertussis pathogenesis have identified an array of cell types as responders to infection with B. pertussis (Higgs et al. 2012) and, here, we will focus on the neutrophil. Pathology specimens from the respiratory tract of human infants who have died of pertussis reveal a background of severe alveolar damage accompanied by varying amounts of neutrophilic infiltration (Paddock et al. 2008; Sawal et al. 2009). Multiple factors contribute to fatalities in pertussis, including

secondary infections, and it is difficult to determine from these specimens how neutrophils contribute to clearance of B. pertussis. Because death from pertussis is not common (Clark 2014), it is also unlikely that these specimens reflect the processes involved in ‘typical’ pertussis which is a chronic, non-fatal, nonpurulent, cough illness. Although experimental infections of mice with B. pertussis do not replicate some aspects of human disease, they have been critical for asking questions that cannot be addressed with human specimens. For example, the bacterial colony counts from lungs of neutropenic mice 14 days after infection with B. pertussis are not greater than those in control mice (Kirimanjeswara et al. 2005; Andreasen and Carbonetti 2009). In contrast, in immunized or convalescent mice, neutropenia severely impairs clearance. These findings raise questions, such as (1) what B. pertussis

Received: 29 July 2015; Accepted: 29 September 2015  C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]

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virulence factors influence killing by neutrophils? (2) what prevents neutrophils from clearing B. pertussis during infection of a na¨ıve mouse? and (3) what changes during the course of an infection with B. pertussis such that neutrophils ultimately play a role in clearance?

What B. pertussis virulence factors influence killing by neutrophils? Pertussis results from the combined activity of an array of virulence factors, some of which interact with neutrophils (Table 1), none of which is more incapacitating toward the neutrophil than the adenylate cyclase toxin (ACT). After toxin is secreted and released from B. pertussis, it binds to host cells and translocates its adenylate cyclase catalytic domain across the plasma membrane to generate supraphysiologic levels of intracellular cAMP (Gray et al. 2004). As little as 10 ng/ml of ACT inhibits the oxidative burst of neutrophils (Confer and Eaton 1982; Eby, Gray and Hewlett 2014) and cAMP levels correlate with inhibition of the oxidative burst (Pearson et al. 1987). ACT also inhibits phagocytosis, chemotaxis and the formation of neutrophil extracellular traps (NETs) (Confer and Eaton 1982; Friedman et al. 1987; Eby et al. 2014). Inhibition of NET formation by ACT is attributable to cAMP-mediated suppression of reactive oxygen species generation which is required for induction of NET release under most circumstances (Brinkmann and Zychlinsky 2012; Yipp and Kubes 2013; Eby et al. 2014). Furthermore, ACT inhibits neutrophil apoptosis—surprising since inhibition of NET formation results in apoptosis under other conditions (Remijsen et al. 2011; Eby et al. 2014). The ability of ACT to affect phagocyte function is attributable to expression of the integrin CD11b/CD18 (CR3), the primary receptor for ACT, which is highly expressed by neutrophils (Guermonprez et al. 2001). ACT binds to oligosaccharide domains in the membrane-proximal C-terminal region of CR3, a component of the receptor that is

associated with cell activation, immunomodulation and clustering with other receptors (Zhou et al. 1993; Thornton et al. 1996; Xia et al. 1999; Ross 2002; Morova et al. 2008; O’Brien et al. 2012; Hasan et al. 2015); however, ACT is not a known agonist of CR3. Like ACT, pertussis toxin (PTX) inhibits neutrophil functions. The active domain of PTX catalyzes ADP ribosylation of the adenylyl cyclase-inhibiting G-protein, Giα . Because the result of PTX’s enzymatic activity is more specific than elevation of cAMP (and resultant depletion of ATP) by ACT, the effects of PTX on neutrophils are less extensive. The f-met-leu-phe (fMLP) receptor, for example, requires functional Giα for downstream signaling and PTX inhibits neutrophil chemotaxis induced by fMLP, but does not inhibit the PMA or zymosan-induced oxidative burst or granule release because these stimulants of chemotaxis do not act through Giα (Becker et al. 1985; Spangrude et al. 1985; Brito et al. 1997; Belisle and Abo 2000). Similarly, PTX inhibits NET formation induced by IL-8 but does not reduce NET formation elicited by PMA or a calcium ionophore (Gupta et al. 2014). PTX is used as a tool to determine if Giα mediates signaling events in neutrophils; the findings of these experiments, while not intended to address pertussis pathogenesis, have identified effects of PTX on signaling related to neutrophil functions, including chemotaxis, oxidative burst, degranulation, cytoskeletal changes and inhibition of apoptosis (Richter, Andersson and Olsson 1989; Belisle and Abo 2000; Gaudreault et al. 2005; Zmijewski et al. 2011; Jung et al. 2013; Pliyev, Ivanova and Savchenko 2014). Many of these studies have implications for the effect of PTX on neutrophils during infection with B. pertussis. Other virulence factors have been demonstrated to interact with neutrophils. Tracheal cytotoxin (TCT) prevents neutrophil migration in response to fMLP (Cundell et al. 1994). Fimbriae bind to neutrophils, but the functional effects of binding are not known (Hazenbos et al. 1995). Filamentous hemagglutinin (FHA) is another B. pertussis surface-expressed molecule with effects on neutrophils. FHA is

Table 1. Bordetella pertussis virulence factors and products that affect neutrophils.

Name

Enzymatic Activity

Binding Target on neutrophils

Adenylate Cyclase Toxin (ACT)

Conversion of ATP → cAMP

Pertussis toxin (PTX)

Function

Reference

CD11b/CD18 (CR3, Mac-1, α M β 2 integrin)

Dysregulation of cellular signaling, consumption of cellular ATP, pore formation

Vojtova, Kamanova and Sebo (2006)

Covalent transfer of ADP-ribosyl group to regulatory GTP-binding proteins

Glycosylated molecules on cell surfaces

Dysregulation of cellular signaling

Locht, Coutte and Mielcarek (2011)

Filamentous hemagglutinin (FHA)

None identified

Possibly CD11b/CD18; additional integrin co-receptors such as LRI/IAP

Attachment, dysregulation of host immune defenses

Villarino, Osicka and Sebo (2014)

Pertactin (PRN)

None identified

Not defined

Dysregulation of host immune defenses, adhesion

Inatsuka et al. (2010); Melvin et al. (2014)

Lipooligosaccharide (BpLOS)

None identified

CD14/TLR4/MD2 Complex

Activation of immunodefense

Marr et al. (2010a)

Tracheal cytotoxin (TCT)

None identified

Not defined

Impairs innate immunodefense cell function

Fedele, Bianco and Ausiello (2013)

Eby et al.

required for attachment of B. pertussis to neutrophils, and interaction of FHA with neutrophils increases the surface expression of CR3, the primary receptor responsible for attachment and phagocytosis of B. pertussis in the absence of opsonizing antibodies (Weingart and Weiss 2000; Mobberley-Schuman and Weiss 2005). Initial experiments implicated both the glycan-binding component and the RGD site on FHA as interacting with CR3 on macrophages (Relman et al. 1990); however, recent studies, employing mutations in the RGD segment and anti-FHA antibodies for blocking binding, have instead suggested that the mature C-terminal domain of FHA is the binding segment (Julio et al. 2009). While the RGD component may not associate with CR3, it may bind to the leukocyte-response integrin (LRI)/IAP complex (α 5 β 3 or CD51/CD61 with CD47) which then upregulates CR3 for enhanced bacterial attachment (Ishibashi, Claus and Relman 1994). Attachment of bacteria to neutrophils, mediated by FHA, may provide benefits to the bacterium. Close approximation of bacteria to neutrophils through FHA-mediated attachment increases the local concentration of ACT (and expression of ACT’s receptor, CR3) which inhibits phagocytosis (Gray et al. 2004; MobberleySchuman and Weiss 2005; Eby et al. 2013). Using conditions under which phagocytosis of B. pertussis does occur, Lamberti et al. (2008) have found that a small fraction of internalized bacteria is not killed within 2 hours by neutrophils and this intracellular population of bacteria has been hypothesized to serve as a reservoir for chronic infection. Whether intracellular survival within neutrophils is of clinical significance has not been determined but is unlikely given the small fraction of surviving organisms compared to the total internalized (Lenz, Weingart and Weiss 2000) and the short lifespan of neutrophils. Several studies have asked whether CR3-mediated attachment and phagocytosis is less effective than antibody-mediated phagocytosis (Saukkonen et al. 1991; Hellwig et al. 2001). Hellwig et al. used multivalent antibodies which allowed crosslinking of B. pertussis to Fc-receptors or CR3 in vivo in mice. Addition of antibodies that crosslinked B. pertussis to CR3 resulted in less efficient phagocytosis than crosslinking antibodies to FCγ RIII/II (Hellwig et al. 2001). It is difficult to interpret these data since crosslinking does not represent a natural interaction with CR3, and crosslinking with Fc-receptors does not preclude CR3mediated interactions. In a follow-up study, which provided data to support the concept that phagocytosis via FCγ R is more efficient than by CR3, B. pertussis was opsonized with human serum spiked with pooled anti-B. pertussis immune IgG and combined with neutrophils in vitro. In the setting of anti-B.pertussis IgG, the phagocytosis and respiratory burst were more robust than without IgG (Rodriguez et al. 2001). Furthermore, intracellular survival in neutrophils is greater (up to 4.8%) when uptake occurs preferentially through CR3 rather than Fc-receptors (Lamberti et al. 2008). Weingart et al. explored attachment and phagocytosis of B. pertussis as well, finding that a greater percentage of bacteria attach to neutrophils in the absence of immune serum than in the presence of serum (Weingart and Weiss 2000; Weingart et al. 2000). This suggested either that attachment via CR3 is more effective than via FCγ R or that immune serum contains anti-FHA antibodies which interfere with FHA-mediated binding to CR3. In fact, depletion of anti-FHA antibodies from immune serum increases attachment (Mobberley-Schuman, Connelly and Weiss 2003). Importantly, ACT blocks phagocytosis initiated by both receptors and phagocytosis is only efficient when ACT is inhibited (Weingart and Weiss 2000; Weingart et al. 2000; Mobberley-Schuman et al. 2003). For these studies, bacteria were centrifuged into apposition with neutrophils, a process that in-

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creases the percentage of attachment and internalization, and also increases the local ACT concentration at the bacterium– neutrophil interface (Lenz et al. 2000; Gray et al. 2004; Eby et al. 2013). These in vitro studies of neutrophil phagocytosis of B. pertussis highlight the powerful ability of ACT to inhibit phagocytosis, and the complexity of the interactions amongst ACT, FHA and host cell receptors. As with attachment and phagocytosis, studies examining neutrophil-mediated killing of B. pertussis in vitro are influenced by the experimental conditions. Lenz et al. (2000) added B. pertussis to neutrophils at a 6:1 multiplicity of infection (MOI) with 0.25% bovine serum albumin, and then centrifuged the bacteria together; they found that 98.3% of added bacteria were killed. Rodriguez et al. (2001) added IgA- or IgG-opsonized B. pertussis to neutrophils at a 70:1 MOI in 10% fetal calf serum without centrifugation and found that 70–85% of added B. pertussis were killed. Ross et al. (2013) found that mouse neutrophils incubated at an MOI of 1:1 with B. pertussis while shaking in the presence of IL-17A or IFN-γ and 10% autologous, convalescent serum killed 60–70% of B. pertussis. IL-17A increased killing in comparison to control neutrophils. While it is possible that the high percentage of killing observed by Lenz et al. is significantly different from the others, and this may be attributable to centrifuging neutrophils together, it is difficult to determine if there are meaningful differences amongst the experiments. Furthermore, all experiments examining neutrophil killing of B. pertussis have been performed with B. pertussis which were washed immediately prior to combining with neutrophils. ACT is not rapidly secreted, and adequate time for secretion prior to combining with neutrophils is required in order to allow enough ACT to be secreted to mimic in vivo concentrations, which can exceed the equivalent of 100 ng/ml of purified, recombinant ACT (Eby et al. 2013). Because many in vitro experiments do not allow time for ACT secretion, it is unclear whether they reflect outcomes of infection with B. pertussis. Regardless of the variability in percent survival and the dependence of the findings on the experimental conditions, B. pertussis can be killed efficiently by neutrophils in vitro.

What prevents neutrophils from clearing B. pertussis during infection of a na¨ıve mouse? In the lungs of mice inoculated by intranasal or aerosol administration of B. pertussis, the number of B. pertussis increases to a peak between days 5 and 7, and then gradually declines over 4– 5 weeks (Table 2) (McGuirk et al. 1998; Harvill, Cotter and Miller 1999; McGuirk and Mills 2000; Kirimanjeswara et al. 2005; Mann et al. 2005). Although there may be a detectable neutrophil response within 1 day after bacterial challenge, there is not a substantial influx of neutrophils into the lungs until day 3, with a peak at day 7. This represents a relatively slow response compared with that elicited by infection with some other Gramnegative respiratory pathogens (Ye et al. 2001; Wang et al. 2002). Based on studies in which neutrophil counts were followed through the duration of B. pertussis infection, the neutrophilic infiltrate decreases in magnitude as bacteria are cleared (see Graphical Abstract). Several studies have focused on how the cytokine and chemokine responses to infection with B. pertussis account for the delayed neutrophil influx into the respiratory tract. After inoculation of mice with B. pertussis, there is a rapid rise (within 6 hours) in the neutrophil-recruiting chemokines KC, MIP-2 and LIX (the murine homologs of the human IL-8 [CXCL8]) (Higgins et al. 2003; Andreasen and Carbonetti 2008). These mediators are

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Table 2. Murine lung bacterial and neutrophil counts over the course of infection with B. pertussis. Inoculation method (bacterial CFU)

Neutrophil collection method

McGuirk et al. (1998); McGuirk and Mills et al. (2000)

Aerosol

Harvill, Cotter and Miller (1999)

Neutrophil elevation, peak, duration

Mouse type

Bacterial strain

CFU peak, duration

BAL

8–12 week BALB/c

Wellcome 28

5–7 days, 35 days

7 days, 14 days, 35 days

i.n. (5 × 10e5)

n/a

4 week BALB/c

Tohama I

7 days, 28 days

n/a

Kirimanjeswara et al. (2005)

i.n. (5 × 10e5)

Homogenized lung

C57BL/6

BP536

3 days, >14 days

n/a, 10 days, >14 days

Gueirard et al. (1998)

i.n. (1.5 × 10e6)

BAL

3–4 week BALB/c

BP18323

7 days, >21 days

n/a

Carbonetti et al. (2005)

i.n. (2.5 × 10e5)

BAL

BALB/c

Tohama I

7 days, 21 days

3 days, n/a, n/a

Moreno et al. (2013)

i.n. (5 × 10e7)

BAL

C3H/HeN

Tohama I

n/a, n/a

1 day, n/a, n/a

Wolfe et al. (2007)

i.n. (5 × 10e5)

Homogenized lung

4–6 week C57BL/6

BP536

n/a, >14 days

n/a, 3 days, n/a

Reference

released in response to Toll-like receptor-4 (TLR4) activation (Andreasen and Carbonetti 2008), and infection of TLR4-deficient mice with B. pertussis, in comparison to wild-type mice, results in delayed MIP-2, delayed neutrophil infiltration, and greater bacterial numbers (CFU) over the course of infection (Higgins et al. 2003; Mann et al. 2005; Moreno et al. 2013). Although Bordetella pertussis lipo-oligosaccharide (BpLOS) does not stimulate cytokine release via TLR4 as effectively as either the LPS of Escherichia coli or the LPS of the related B. bronchiseptica, bacterial counts from the lungs of TLR4-deficient mice infected with B. pertussis are greater than counts from the lungs of control mice on days 7 and 14 (Mann et al. 2005; Fedele et al. 2008; Marr et al. 2010b). Thus, TLR4-mediated signaling contributes to clearance of infection with B. pertussis in mice. Importantly, human TLR4 is less sensitive to LOS than murine TLR4 (Marr et al. 2010a), and data regarding LOS-mediated neutrophil recruitment gathered from the mouse model should be interpreted with this in mind. Despite activation of pathways for neutrophil recruitment in the na¨ıve mouse within 6 hours after B. pertussis challenge, neutrophil influx into the airway does not occur for another 1–2 days. In contrast, infection with a PTX-deletion mutant of B. pertussis results in an earlier chemokine response of greater magnitude, earlier neutrophil influx and lesser burden of bacteria throughout the course of infection compared with wild-type B. pertussis (Carbonetti et al. 2003; Andreasen and Carbonetti 2008). This effect is consistent with disruption of TLR4-mediated signaling by PTX (Zhang and Morrison. 1993). In addition, studies performed primarily in vitro show that PTX inhibits fMLP-, zymosan- and LPS-stimulated neutrophil chemotaxis (Becker et al. 1985; Spangrude et al. 1985; Brito et al. 1997). Thus, inhibition of neutrophil chemotaxis by PTX can occur at multiple levels, as supported by both in vitro and in vivo findings. Although an early neutrophil response to B. pertussis may be suppressed by PTX, neutrophils eventually do respond to infection in mice, and the response is mediated, at least in part, by IL-17. Bordetella pertussis infection of mice induces IL-17 gene expression in lung tissue and B. pertussis stimulates IL-17 expression in cells from lungs and spleens at ∼7 days post-infection

(Banus et al. 2008; Andreasen, Powell and Carbonetti 2009; Dunne et al. 2010; Connelly, Sun and Carbonetti 2012; Ross et al. 2013). Neutralization of the IL-17 response to B. pertussis infection by administration of an anti-IL-17 antibody blocks KC elevation and subsequent neutrophil recruitment (Andreasen, Powell and Carbonetti 2009). Similarly, in IL-17 knockout mice, neutrophil recruitment is reduced and delayed (Ross et al. 2013) and challenge with B. pertussis results in a greater bacterial burden during the later stages of infection (>day 14) compared with wild-type mice (Dunne et al. 2010; Zhang et al. 2011b; Ross et al. 2013). Collectively, these data strongly suggest that the IL-17-mediated recruitment of neutrophils is important for late clearance of infection from na¨ıve mice. Cytokines that stimulate the IL-17 response, such as IL-1β and IL-6, are also important for control of B. pertussis infection. The signaling receptor for IL-1β and IL-1α is IL-1R, Type 1 (IL-1R1)(Dinarello 2013), and IL-1R1 -/- mice exhibit reduced ability to generate an IL-17 response during B. pertussis infection and impaired clearance of the bacteria compared with wildtype mice (Dunne et al. 2010). The increased bacterial burden in IL-1R1 -/- mice is not, however, associated with decreased neutrophil recruitment; rather the neutrophil influx is greater after day 5 in comparison to control mice (Zhang et al. 2011b). Whether this delayed but enhanced inflammation is attributable to the increased bacterial load or other factors has not been determined. In contrast, the effect of IL-6 on the IL-17 response to B. pertussis is more easily defined. Relative to na¨ıve wild-type mice, na¨ıve IL-6 -/- mice challenged with B. pertussis exhibit decreased IL-17 response, decreased MIP-2 and LIX, decreased neutrophil recruitment, increased late bacterial numbers and prolonged duration of infection (Zhang et al. 2011a). In summary, a critical driver of neutrophil recruitment, particularly during the later phase of B. pertussis clearance, is the IL-17 response and associated chemokine stimulation. Studies in mice suggest that Th17 cells are a source of the IL-17 response that promotes late B. pertussis clearance. Bordetella pertussis infection results in generation of antigen-specific Th17 cells (Banus et al. 2008; Dunne et al. 2010; Ross et al. 2013). IL-6 and IL-1 knockout mice are impaired in their ability to

Eby et al.

generate the antigen-specific response (Dunne et al. 2010; Zhang et al. 2011a). In addition, infection of nude mice (deficient in T lymphocytes) or SCID mice (deficient in both T cells and B cells) with B. pertussis results in a persistent elevation of bacterial counts in the mouse lung, and the difference in bacterial counts between wild-type and lymphocyte-deficient mice becomes evident after ∼1–2 week of infection (Mills et al. 1993; Barbic et al. 1997). This timing is coincident with the IL-17mediated neutrophil influx in wild-type mice, consistent with a role for T cells in the phenomenon, although no study has examined neutrophil influx in lymphocyte-deficient mice. There are other potential sources of IL-17, including neutrophils, natural killer T cells and γ δ T cells (Cua and Tato 2010) and the role of these cell types in the B. pertussis-associated IL-17 response has not been clearly addressed. Neutrophils from the lungs of mice infected with B. pertussis are positive for IL-17 by intracellular immunocytochemical staining, but it is not possible to determine if this is attributable to secretion of IL-17 or binding of IL-17 to the receptors of these cells (Andreasen, Powell and Carbonetti 2009). In summary, while there is evidence for Th17-produced IL-17 in the response to B. pertussis, the function of other cell types in the IL-17 response has yet to be determined. Multiple B. pertussis virulence factors contribute to eliciting the IL-17 response. Four days after challenge of mice with PTX-negative B. pertussis, there is less IL-17 gene expression in lungs in comparison to wild-type B. pertussis (Andreasen, Powell and Carbonetti 2009). These findings suggest that PTX promotes an IL-17 response starting several days after inoculation, in contrast to its initial suppression of neutrophil recruitment. Consistent with these in vivo findings, PTX stimulates bone marrow-derived macrophages to produce IL-1β in vitro (Zhang et al. 2011b). In addition to PTX, ACT stimulates production of IL-1β from bone marrow-derived dendritic cells and generation of antigen-specific Th17 cells in a manner dependent on IL-1R1 expression (Dunne et al. 2010). Finally, BpLOS induces monocytederived dendritic cells to produce IL-23, IL-6 and IL-1β that result in IL-17 production from na¨ıve T cells (Fedele et al. 2008, 2010). Stimulation of IL-17 through TLR4 signaling is consistent with induction of IL-17 responses by other pathogens such as Klebsiella pneumoniae (Happel et al. 2003; McKenzie, Kastelein and Cua 2006). Thus, while it would seem beneficial for the bacterium to inhibit neutrophil influx, several of its virulence factors promote the neutrophil-recruiting IL-17 response that starts to mature ∼7 days after inoculation. Investigators have hypothesized that the inflammatory response to PTX, including the increase in IL-17, may promote cough and transmission (Andreasen, Powell and Carbonetti 2009; Connelly et al. 2012). While the cough is a defining characteristic of pertussis, mice do not exhibit cough after infection, nor do they reproducibly transmit pertussis. Infection of non-human primates with B. pertussis replicates clinical and immunological aspects of human disease and studies in this model have addressed the IL-17 response (Warfel et al. 2012; Warfel, Beren and Merkel 2012). After inoculation of baboons with B. pertussis, nasopharyngeal washes (NPW) contain IL-6, IL-23 and IL-1β, followed by IL-17 at 5–7 days after infection (Warfel and Merkel 2013). On days 9 and 10, IL-8 is elevated in the NPWs, consistent with chemokine induction by IL-17. The IL-17 response in these baboons is long lasting, with peripheral blood mononuclear cells exhibiting B. pertussisspecific IL-17 release for 24 months (the latest point tested) (Warfel and Merkel 2013). Thus, the findings in na¨ıve mice regarding the IL-17 response to B. pertussis infection are supported by the non-human primate model.

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What changes during the course of an infection with B. pertussis such that neutrophils ultimately play a role in clearance? During the first few days of infection of a na¨ıve mouse, the neutrophil is not able to contribute to clearing B. pertussis largely because PTX prevents neutrophil recruitment to the airway. However, even when mice are infected with PTX B. pertussis, allowing neutrophils to immediately move into the airway in response to B. pertussis, there is no difference in burden of infection between neutropenic and non-neutropenic mice at day 7 or 14 (Andreasen and Carbonetti 2009). This could mean that the other bacterial virulence factors that impair neutrophil functions are active in the absence of PTX, additional host factors are needed to assist the neutrophil, or both. In convalescent or vaccinated mice, neutrophils play an important role in clearing infection, suggesting that antibody-mediated clearance, through a combination of opsonization and virulence-factor neutralization, is able to overcome the inhibition of neutrophils at the site of infection (Andreasen and Carbonetti 2009). This concept is supported by the finding that transfer of antibodies from convalescent mice promotes clearance of B. pertussis from mouse lungs, and that this clearance is abrogated by neutrophil depletion or FCγ receptor deletion (Kirimanjeswara et al. 2005). The clearance of B. pertussis enabled by convalescent antibodies occurs within the first 3 days after inoculation if mice are infected with a PTX B. pertussis, but later in infection with wild type. These findings regarding the importance of antibodies in clearance may explain, in part, the clearance of B. pertussis during the late stages of infection in a na¨ıve mouse (>day 14). Antibodies to B. pertussis are detectable between days 14 and 24 after infection, and bacterial counts from lungs in IgG -/- mice remain persistently elevated with a difference in counts starting around day 14 (Mahon et al. 1997). Thus, whether provided by prior infection, development of an antibody response in a na¨ıve mouse, antibody transfer or vaccination, antibodies to B. pertussis promote clearance of infection in conjunction with neutrophils. Certainly, additional cell types and factors may promote clearance. For example, IL-17 not only mediates neutrophil recruitment but increases their ability to kill B. pertussis (Ross et al. 2013). Findings regarding IL-17 could influence the development of new B. pertussis vaccines. While all cell types contributing to the IL-17 response during B. pertussis infection have not been identified, there is a lasting antigen-specific Th17 response in mice and baboons (Dunne et al. 2010; Zhang et al. 2011a; Warfel and Merkel 2013). Like the directed memory Th17 responses generated by infection in baboons, the whole cell pertussis vaccine (wP) also stimulates an antigen-specific Th17 response that contributes to protection in mice (Higgins et al. 2006; Banus et al. 2008). The candidate, live-vaccine strain of B. pertussis, BPZE1, which expresses ACT, detoxified PTX, BpLOS and FHA, also generates a Th17 response in mice (Kammoun et al. 2012). While both wP and BPZE1 contain LOS which may promote an IL-17 response via TLR4 stimulation, an alum-containing acellular pertussis vaccine generates IL-17-mediated protection in mice even though it contains essentially no LOS (Ross et al. 2013). Although a Th17 response to aP may be particular to mice and has not been reported under all conditions (Banus et al. 2008), the presence of LOS is not required to elicit IL-17. The Th17 response to vaccines is balanced by either Th2 (aP) or Th1 (wP or natural infection of na¨ıve host) responses (Ross et al. 2013). Design of an optimal vaccine likely requires the appropriate balance between Th1 and Th17 lymphocyte responses for the combination

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of high efficacy and low risk of side effects. Novel adjuvants are being developed to accomplish this goal (Libster and Edwards 2012; Polewicz et al. 2013; Meade, Plotkin and Locht 2014; Plotkin 2014; Dunne et al. 2015 ). Ultimately, promoting the effectiveness of neutrophils against B. pertussis should be approached not just from the perspective of neutralizing virulence factors that suppress their functions but also through stimulating the immune response that enhances their recruitment and activity. Investigation into the signaling mechanisms and pathways underlying neutrophil recruitment and function in the setting of B. pertussis has improved our knowledge of disease pathogenesis and vaccine design. Substantial gaps remain, however, in the understanding of the neutrophil’s role in controlling infection with B. pertussis. What additional factors contribute to the early cytokine and chemokine response? Can further studies shed light on the mechanisms by which IL-1 and IL-6 contribute to neutrophil-mediated clearance of B. pertussis? Which cell types, in addition to T lymphocytes, are responsible for secreting IL-17 during infection? What accounts for the opposing functions of PTX as both a neutrophil inhibiting and recruiting toxin? What promotes bacterial clearance in the late phase of B. pertussis infection? The study of neutrophil-mediated defense against B. pertussis infection will continue to yield important information about pathogenesis and has potential to provide generalizable information about the crosstalk between the adaptive and innate arms of the immune system.

FUNDING This work was supported by the National Institutes of Health [HHSN272201200005C-416476 to J.C.E., 5T32A1007046-38 to C.L.H., and 5 R01 AI1018000, HHSN272201200005C-416477 to E.L.H.]. Conflict of interest. None declared.

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