Mast cells inhibit intramacrophage Francisella tularensis replication via contact and secreted products including IL-4 Jyothi M. Ketavarapu*, Annette R. Rodriguez*, Jieh-Juen Yu*, Yu Cong*, Ashlesh K. Murthy*, Thomas G. Forsthuber*, M. Neal Guentzel*, Karl E. Klose*, Michael T. Berton†, and Bernard P. Arulanandam*‡ *South Texas Center for Emerging Infectious Diseases and Department of Biology, University of Texas, San Antonio, TX 78249; and †Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, TX 78229
Francisella tularensis is an intracellular, Gram-negative bacterium that is the causative agent of pulmonary tularemia. The pathogenesis and mechanisms related to innate resistance against F. tularensis are not completely understood. Mast cells are strategically positioned within mucosal tissues, the major interface with the external environment, to initiate innate responses at the site of infection. Mast cell numbers in the cervical lymph nodes and the lungs progressively increased as early as 48 h after intranasal F. tularensis live vaccine strain (LVS) challenge. We established a primary bone marrow-derived mast cell–macrophage coculture system and found that mast cells significantly inhibit F. tularensis LVS uptake and growth within macrophages. Importantly, mice deficient in either mast cells or IL-4 receptor displayed greater susceptibility to the infection when compared with corresponding wild-type animals. Contact-dependent events and secreted products including IL-4 from mast cells, and IL-4 production from other cellular sources, appear to mediate the observed protective effects. These results demonstrate a previously unrecognized role for mast cells and IL-4 and provide a new dimension to our understanding of the innate immune mechanisms involved in controlling intramacrophage Francisella replication. innate 兩 LVS 兩 macrophages 兩 mucosal 兩 pulmonary
F
rancisella tularensis is a Gram-negative facultative intracellular bacterium that causes the life-threatening disease ‘‘tularemia’’ in humans (1). Because of the high degree of infectivity through the aerosol route (2, 3), F. tularensis has been considered a potent biological weapon (4). F. tularensis is classified into several subspecies including the most relevant to human disease: Francisella tularensis subspecies tularensis (type A) and subspecies holarctica (type B) (3). The F. tularensis live vaccine strain (F. tularensis LVS, derived from type B strain) is attenuated for virulence in humans and has been used for immunization (5). To date, most studies have used LVS infection in mice to examine immune defenses against Francisella infection (6). Early production of IFN-␥ and TNF-␣ (7–9) are required for protection against primary LVS infection. There is evidence to suggest that different host defense mechanisms may be required to clear a systemic versus pulmonary LVS infection (10, 11). Within the respiratory system, distinct mechanisms contribute to innate defenses in the airways and alveolar spaces. Mucociliary clearance predominates in the airways, whereas phagocytic cells such as macrophages and neutrophils are involved in removal of microorganisms that reach the deeper alveolar spaces (12). The activation of toll-like receptor (TLR)-2 and MyD88-dependent signaling in phagocytic cells has been reported recently to be important in early defenses against Francisella as evidenced by increased bacterial burden and enhanced susceptibility in knockout animals (13–15). Macrophages are the primary host cells for Francisella replication (1), and the interactions of this cell type with others in the control of intracellular growth have yet to be fully characterized. www.pnas.org兾cgi兾doi兾10.1073兾pnas.0707636105
Mast cells have long been recognized as the primary cells involved in allergic conditions associated with type I hypersensitivity reactions (16, 17). There is accumulating evidence to suggest that mast cells contribute and shape innate and adaptive defenses through the production and release of preformed and de novo-synthesized mediators such as histamine, leukotrienes, prostaglandins, and a host of chemokines and cytokines, including TNF-␣ and IL-4 (18, 19). Mast cells also have been shown to directly phagocytose adherent microorganisms (20, 21) and to use oxidative means to kill bacteria in an opsonin-dependent fashion (21, 22). Importantly, mast cells are strategically positioned in the lungs to initiate innate responses at this site of infection (23) and to selectively migrate into regional lymph nodes to influence the development of adaptive immunity (18). Specifically, mast cells have been demonstrated to be important in mediating protective immunity against Gram-negative and Mycoplasma-induced pneumonias (24–27). The extensive phenotypic plasticity of mast cells, their proximity to primary sites of infection, and their potential to influence important aspects of the immune response, all likely contribute to their function in the immune response to bacterial infection. As such, these cells may play an underappreciated role in innate defenses against respiratory tularemia. In this study, we used a bone marrow-derived mast cell– macrophage coculture system and found that mast cells inhibit bacterial growth within primary macrophages by contactdependent mechanisms and secreted products including IL-4. Mice deficient in either mast cells or IL-4 receptor displayed greater susceptibility to the infection than corresponding wildtype animals. Collectively, these results are indicative of a previously unrecognized pathway that may be important in the alternative activation of macrophages for host resistance against pneumonic tularemia. Results Mast Cell Infiltration into the Respiratory Compartment after Intranasal (i.n.) F. tularensis LVS Challenge. We analyzed the cellular
infiltration into the cervical lymph nodes that preferentially drain the oropharyngeal compartment (28), and into the lungs, which serve as the primary site of infection (1), in C57BL/6 mice after i.n. F. tularensis LVS (3,000 cfu, or ⬇1 LD50) challenge. At 24, 48, and 72 h after challenge, lung and cervical lymph node Author contributions: B.P.A. designed research; J.M.K., A.R.R., J.-J.Y., Y.C., and A.K.M. performed research; T.G.F., K.E.K., and M.T.B. contributed new reagents/analytic tools; J.M.K., A.R.R., M.N.G., and B.P.A. analyzed data; and B.P.A. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. ‡To
whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/ 0707636105/DCSupplemental. © 2008 by The National Academy of Sciences of the USA
PNAS 兩 July 8, 2008 兩 vol. 105 兩 no. 27 兩 9313–9318
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Edited by Roy Curtiss, Arizona State University, Tempe, AZ, and approved April 24, 2008 (received for review August 13, 2007)
Fig. 1. Mast cell-dependent inhibition of F. tularensis LVS replication in macrophages. (A) Mast cells, macrophages, and cocultures were infected with LVS. Bacterial counts at 3 and 24 h after challenge with MOI of 10 or 100 of LVS. *, P ⫽ 0.004 (two-way ANOVA). ⽤, P ⫽ 0.002 (two-way ANOVA). (B) Bacterial counts in cell lysates and respective supernatants at 2 and 24 h after challenge with LVS (MOI 10). *, P ⬍ 0.001 (unpaired t test) macrophages versus mast cell–macrophage coculture. Results are representative of three independent experiments.
cells were analyzed by flow cytometry for infiltration of inflammatory cells (macrophages, CD11b; dendritic cells, CD11c; neutrophils, Ly6Gr1; and mast cells, FcRI). These analyses revealed little early (24 h) infiltration of inflammatory cells into the cervical lymph nodes and lungs, except an elevated frequency of neutrophils in the cervical lymph nodes, after i.n. compared with mock (PBS) LVS challenge (supporting information (SI) Fig. S1 A and B). By 48 h, frequencies of all of the analyzed cell types were elevated in cervical lymph nodes (Fig. S1 A), but only mast cell frequency was enhanced in the lungs (Fig. S1B). Greater than 95% of the FcRI⫹ cells were c-Kit⫹ confirming the identity as mast cells (data not shown). At 72 h, there was a trend towards increases in each examined cell type in both cervical lymph nodes (Fig. S1 A) and lungs (Fig. S1B). These analyses demonstrate that, apart from conventional phagocytic cells such as macrophages and neutrophils, mast cells infiltrate early into the sites of pulmonary infection after F. tularensis LVS challenge. Additionally, mast cells appear to infiltrate the lungs of infected animals earlier (48 versus 72 h after challenge) than other examined cell types. Mast Cell-Induced Inhibition of F. tularensis LVS Replication in Macrophages. Because mast cells are observed at the site of
infection with other known Francisella permissive cell types such as macrophages (1), we examined the interaction of mast cells and macrophages with F. tularensis LVS by using a primary bone marrow-derived cell culture system. Mast cells (98% FcRI⫹), macrophages (96% CD11b⫹), or combined cocultures were incubated for 2 h with LVS [multiplicity of infection (MOI) of either 10 or 100], followed by gentamicin treatment for 1 h to kill extracellular bacteria. The cells were then washed, cultured in fresh media, lysed at 3 or 24 h after challenge and bacterial replication was determined by dilution plating. As shown in Fig. 1A, there was limited replication of LVS within mast cells in contrast to the robust replication observed in macrophage cultures. Specifically at 24 h, there was a 2.6 – 4 log10 (P ⫽ 0.004) increase in intracellular bacteria in macrophages infected at the two MOIs, compared with only a 0.5–1 log10 increase in mast cells. Interestingly, LVS replication at 24 h was reduced by 3– 4 log10 (⬇98% inhibition) at the two MOIs within the macrophage–mast cell cocultures (P ⫽ 0.002), when compared with macrophages in the absence of mast cells. To address whether the reduced recovery in mast cells and mast cell–macrophage cocultures was caused by reduced bacterial uptake or replication, we analyzed both the supernatants and cell lysates of the different cell culture conditions at 2 h after incubation with LVS (no gentamicin treatment). As shown in Fig. 1B, mast cells alone (3.5E⫹2 ⫾ 1.25E⫹2 cfu) and mast 9314 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0707636105
cell–macrophage cocultures (1.57E⫹3 ⫾ 1.5E⫹2) displayed significantly reduced (⬇2 log10 and ⬇1.5 log10, respectively) intracellular bacteria at 2 h compared with macrophages alone (1.4E⫹4 ⫾ 2.1E⫹3). In contrast, the supernatant fluids at 2 h from the mast cells alone (2.45E⫹7 ⫾ 1E⫹6), and mast cell– macrophage cocultures (3.75E⫹7 ⫾ 3.5E⫹6) contained significantly more bacteria than macrophages alone (1.8E⫹6 ⫾ 5.5E⫹3). Collectively, these results suggest that mast cells have reduced uptake of LVS and that they reduce uptake into macrophages in coculture. Additionally, to determine whether the lower numbers of intracellular bacteria observed in mast cells and cocultures compared with macrophages at 24 h was caused by an early extrusion of bacteria, we enumerated LVS in both supernatant and cell lysates at this time point. At 24 h, the number of intracellular bacteria in mast cells alone and in mast cell–macrophage cocultures increased to a lower extent (⬇1 log10) than in macrophages alone (⬇2 log10) compared with the counts at 2 h in the corresponding cultures. Minimal amounts of LVS were recovered from each of the supernatants at 24 h. Collectively, these results suggest that the reduced bacterial numbers at 24 h postchallenge in mast cells and mast cell– macrophage cocultures, compared with macrophages alone, was caused by a combination of early reduced uptake and inhibition of intracellular replication, but not by extrusion of replicated bacteria from cultures with mast cells. Mast Cell-Mediated, Contact-Independent Inhibition of Intramacrophage LVS Replication. To determine whether the effect of mast cells
on F. tularensis LVS replication within macrophages requires direct mast cell–macrophage contact, a transwell system was established. Specifically, mast cells and macrophages were cultured in separate chambers within the transwell to avoid direct contact. At 24 h, the cells were lysed to determine bacterial counts. There was a 2 log10 (P ⫽ 0.002) decrease in LVS recovered from macrophages cocultured with mast cells within the transwells, compared with macrophages cultured in the absence of mast cells (Fig. 2A). There were no significant differences between bacterial replication in mast cells within the transwell system, whether cultured alone or in the presence of macrophages. These results suggest that the mast cell-induced inhibition of intramacrophage LVS replication in direct coculture (3–4 log10 inhibition) could be reproduced in transwells without direct contact (2 log10 inhibition), albeit not completely, suggesting that both contact-dependent and contact-independent events contribute to the inhibitory effect. To examine further the possibility that the inhibition of LVS replication might require a mast cell-derived cytokine, supernatant fluid was collected from mast cells at 24 h after challenge, filtered, and used to culture macrophages infected with LVS. There was a 1.7 log10 (P ⫽ 0.001) decrease in LVS replication in Ketavarapu et al.
macrophages cultured with supernatant from LVS-infected mast cells compared with supernatant from uninfected mast cells (Fig. 2B). In contrast, there was minimal effect on intracellular bacterial replication when supernatants from LVS-infected macrophages were used. Similar results were observed at an MOI of 100 (data not shown). The supernatants from the LVS-infected mast cell cultures were then analyzed for TNF-␣ and IL-4 (Fig. 2C), the primary cytokines produced by mast cells (18). TNF-␣ was detected in culture supernatants of both infected mast cells and infected macrophages, indicating that this was not a mast cell-specific signal. However, IL-4 was found only in the supernatants of LVS-infected mast cells and in cocultures. Interestingly, cocultured LVS-infected cells at an MOI of 100 produced greater amounts of IL-4 than infected mast cells alone, which may be indicative of the ability of macrophages to further cross-activate mast cells (Fig. S2). There was minimal induction of cytokines in mock-infected cultures. Collectively, these results suggest that IL-4 may be an important component of mast cell-secreted products that mediate the inhibition of F. tularensis LVS replication in macrophages. Effect of IL-4 on the Intracellular Replication of F. tularensis LVS. To
examine the direct effect of IL-4 on intracellular replication of LVS, mast cells, macrophages, or mast cell–macrophage cocultures were treated with recombinant (r) IL-4 and infected with LVS (MOI of 10) (Fig. 3A). There was a 3 log10 (P ⫽ 0.0004) reduction of intracellular bacterial growth when rIL-4 was added to LVS-infected macrophages and an additional 1.3 log10 (P ⫽ 0.004) reduction in intracellular bacterial growth in rIL-4-treated macrophage–mast cell cocultures, when compared with the respective untreated infected cells at 24 h. Importantly, inhibition of bacterial replication was abrogated in these cultures on treatment with neutralizing anti-IL-4 antibody. Specifically, treatment with anti-IL-4 antibody along with rIL-4 resulted in an increase of 2.3 log10 (P ⫽ 0.002) in bacterial multiplication within macrophages and a 2.1 log10 (P ⫽ 0.002) increase in cocultures, Ketavarapu et al.
The Contribution of Mast Cells in Innate Defenses Against Pulmonary F. tularensis LVS. We further analyzed the role of mast cells and
mast cell-derived IL-4 in control of pulmonary LVS infection in vivo. To this end, we compared mast cell-deficient (W/Wv) and congenic wild-type (WT) mice challenged i.n. with LVS (3,000 cfu). The frequencies of macrophages, the primary host cell type for Francisella replication, were found to be comparable in W/Wv and WT mice (data not shown). There was an increase in the number of mast cells [detected by naphthol AS-D chloroacetateesterase (25) and morphology] in the cervical lymph nodes of WT mice by 24 and 48 h postchallenge and, as expected, no mast cells were detected in the lymph node sections of W/Wv mice (Fig. 4A). LVS-challenged WT and W/Wv mice also were monitored for survival and weight loss (Fig. 4B). These analyses revealed that W/Wv mice were highly susceptible (20% survival) to pulmonary LVS infection compared with WT animals that exhibited 80% survival to the challenge inoculum (3,000 cfu). Moreover, W/Wv mice experienced progressive weight loss until they succumbed to the infection, in contrast to the WT mice, which showed only a modest and transient weight loss. To determine whether the accelerated mortality observed in the W/Wv mice correlated with enhanced microbial replication, PNAS 兩 July 8, 2008 兩 vol. 105 兩 no. 27 兩 9315
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Fig. 2. Mast cell-mediated contact-independent inhibition of intramacrophage LVS replication. (A) LVS-infected (MOI 10) mast cells and macrophages (top and bottom chambers, respectively) were cultured within transwells for 24 h. *, P ⫽ 0.002 (two-way ANOVA). (B) LVS-infected (MOI 10) macrophages cultured with spent filtered supernatant collected from LVS or mock (PBS) infected mast cells or macrophages. *, P ⫽ 0.001 (two-way ANOVA). (C) TNF-␣ and IL-4 levels in LVS infected culture supernatants. *, P ⫽ 0.002 (two-way ANOVA) 3 h infected cultures versus mast cells. ⽤, P ⫽ 0.0001 (two-way ANOVA) 24 h infected cultures versus mast cells. Results are representative of three independent experiments.
when compared with the respective rIL-4-treated cultures. Furthermore, the addition of neutralizing anti-IL-4 antibody alone to macrophage–mast cell cocultures reversed the inhibition of intracellular bacterial growth. As observed previously, there was limited replication of LVS within mast cells, and this replication was unaffected by rIL-4 or anti-IL-4 treatment. Similar effects were observed when cells were infected at an MOI of 100 (data not shown). These results demonstrate that IL-4 stimulation of macrophages, in the absence of mast cells, can significantly inhibit the intramacrophage replication of F. tularensis LVS. To further support a role for IL-4 in the inhibition of intracellular LVS replication, macrophages and mast cells were isolated from IL-4⫺/⫺ and IL-4⫹/⫹ mice and analyzed for intracellular LVS replication. IL-4⫺/⫺ mast cells were unable to inhibit intracellular bacterial replication when cocultured with IL-4⫺/⫺ macrophages, whereas IL-4⫹/⫹ mast cells in coculture with IL-4⫹/⫹ macrophages inhibited LVS replication (Fig. 3B). There was a 2 log10 (P ⫽ 0.004) greater replication in IL-4⫺/⫺ cocultures compared with IL-4⫹/⫹ cocultures at 24 h. LVS replicated poorly in both IL-4⫹/⫹ and IL-4⫺/⫺ mast cells, whereas the organism replicated well within both sets of macrophages. Moreover, we determined that STAT6-competent mast cells (producing IL-4), but not STAT6-deficient mast cells [abrogated IL-4 production (29)] induced inhibition of LVS replication within wild-type macrophages (Fig. S3). The in vivo relevance of IL-4 in pulmonary LVS infection was examined by determining survival and bacterial burden in BALB/c IL-4R⫺/⫺ and IL-4R⫹/⫹ animals challenged i.n. with LVS (3,000 cfu) (Fig. 3C). These analyses revealed that IL-4R⫺/⫺ animals exhibit greater susceptibility (33% survival) to the pulmonary infection compared with similarly challenged IL4R⫹/⫹ mice (67% survival). To further evaluate microbial replication in these mice, bacterial burdens were measured in the lungs, livers, spleens, and cervical lymph nodes after i.n. LVS challenge (Fig. 3D). By day 3 after LVS challenge, there were significantly greater numbers of recoverable LVS in the lungs (P ⫽ 0.012), spleens (P ⫽ 0.034), and considerable increases in recoverable bacteria from the cervical lymph nodes of the IL-4R⫺/⫺ mice compared with WT animals. On day 6, there were greater numbers of bacteria in the lungs and cervical lymph nodes of IL-4R⫺/⫺ mice compared with WT animals, but the differences were not statistically significant. Together, we have demonstrated a role for mast cell-derived IL-4 in control of intramacrophage bacterial replication, and a function of this cytokine in early defenses against pulmonary LVS infection.
Fig. 3. Effect of IL-4 on the replication of intracellular LVS. Cells were infected with LVS (MOI 10) and cultured in media alone, rIL-4, or neutralizing anti-IL-4 antibody for 24 h. (A) Bacterial replication was determined by dilution plating of lysed cells. *, P ⬍ 0.005 (three-way ANOVA) compared with corresponding untreated and anti-IL-4 antibody-treated cultures, respectively. (B) IL-4⫹/⫹ and IL-4⫺/⫺ mast cells, macrophages, and cocultures were infected with LVS (MOI 10) and cultured in media alone for either 3 or 24 h. *, P ⬍ 0.005 (three-way ANOVA) between IL-4⫹/⫹ and IL-4⫺/⫺ cocultures at 3 and 24 h, respectively. (C) Survival of IL-4R⫺/⫺ and IL-4R⫹/⫹ mice (six animals per group) challenged i.n. with LVS (3,000 cfu). (D) Bacteria recovered from the lungs, liver, spleen, and cervical lymph nodes of IL-4R⫺/⫺ and IL-4R⫹/⫹ mice (four animals per group) on day 3 or 6 after i.n. challenge with LVS. *, P ⱕ 0.03 (two-way ANOVA) compared with WT mice. Results are representative of two to three independent experiments.
bacterial burdens were measured in the lungs, livers, and spleens of W/Wv and WT mice challenged i.n. with LVS (Fig. 4C). By 6 days postchallenge, there was ⬇1,000-fold (P ⬍ 0.001) more bacteria recovered from the lungs and the spleens of the W/Wv mice than from those of the WT animals. These results indicate the importance of mast cells in defense against pulmonary LVS challenge. To evaluate the role of contact-dependent mechanisms versus secreted mast cell products, we treated LVS-challenged WT mice with disodium cromoglycate (DSCG; 10 g/g body weight) to stabilize mast cell membranes (30–32). DSCG-treated mice demonstrated a significantly greater loss of body weight compared with mock (PBS)-treated mice early after pulmonary LVS challenge (3,000 cfu), but progressed to regain the initial body weight by day 14 (Fig. S4 A). As expected, mock (PBS)challenged mice with or without DSCG treatment did not display body weight loss. Moreover, DSCG-treated mice displayed significantly greater lung bacterial burden (P ⬍ 0.001) on day 6 after LVS challenge compared with mock (PBS) treated mice (Fig. S4B). Bacterial burdens within secondary target organs including the liver and spleen were comparable (data not shown). Together, these results suggest that secreted products from mast cells, albeit to a lesser degree than contact-dependent events, contribute to defenses against pulmonary LVS infection. To directly assess whether IL-4 from mast cells was essential in conferring protection against pulmonary LVS challenge (3,000 cfu), bone marrow-derived mast cells were generated from IL-4⫺/⫺ and WT mice and adoptively transferred into recipient W/Wv mice (Fig. S5 A and B). These analyses revealed that transfer of IL-4⫺/⫺ and wild-type mast cells reconstituted equivalent protection (85% survival) in LVS-challenged W/Wv mice, with body weight loss similar to challenged WT mice. As previously seen, W/Wv mice not receiving mast cells exhibited 9316 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0707636105
considerable loss of body weight and succumbed to the bacterial challenge (0% survival), whereas 67% of the WT mice were protected. Based on these analyses, it appears that IL-4 production from mast cells may not be absolutely essential for protection against LVS challenge. The effects of mast cell-derived IL-4, however, may have been masked by contact-dependent effects and non-IL-4-secreted products of mast cells. Additionally, there may be alternative sources of IL-4 production in vivo. To this end, cervical lymph nodes from WT mice displayed enhanced staining for mast cells (green, anti-FcRI; and confirmed by morphology) by day 5 after LVS challenge (Fig. 4D). Moreover, IL-4 (red, anti-IL-4) in the LVS-infected WT lymph node sections was induced and colocalized (yellow) with the mast cells. Whereas there was no staining for mast cells in the W/Wv lymph node sections, there was detectable expression of IL-4 (white arrow) within the W/Wv lymph nodes, albeit at much lower levels than in WT mice, suggesting alternative sources of IL-4 production in vivo. Collectively, contact-dependent events and secreted products of mast cells, including IL-4, appear to play an important role in early defenses against pulmonary LVS challenge. Discussion Mechanisms related to innate resistance and protective immunity against F. tularensis after pulmonary exposure are not completely understood. The ability of this pathogen to effectively subvert or overcome innate defenses thus underscores the need to better understand components of early respiratory defenses that may be involved in limiting intracellular growth and dissemination. By using an in vitro coculture system and a pulmonary challenge model, we now have identified a previously unrecognized contribution of mast cells and their pivotal Ketavarapu et al.
interaction with macrophages in the control of Francisella growth. The early infiltration of mast cells after pulmonary exposure to F. tularensis LVS may allow them to interact with other phagocytic cells, such as alveolar macrophages and dendritic cells, in the respiratory compartment. By using an in vitro system, we found that LVS uptake and replication within macrophages was inhibited on coculture with infected mast cells. Additionally, this inhibition was reproducible by the addition of mast cell-spent supernatants, albeit to a lesser extent than direct coculture, suggesting the contribution of both contact-dependent events and secreted mast cell products to the observed effect. To this end, mast cells are capable of influencing other cells types by utilization of surface structure (e.g., CD40L, FcR1, and MHC) and/or soluble factors such as cytokines (19). We now have determined that IL-4, a cytokine secreted by mast cells, contributed to these inhibitory effects. This was demonstrated by directly using the recombinant cytokine and further validated by using primary cells derived from IL-4⫺/⫺ animals. In this regard, the alternative activation of macrophages by IL-4 (33), apart from the classical inflammatory cytokines IFN-␥ and TNF-␣, has been shown to enhance the bactericidal activity of macrophages against Listeria monocytogenes (34) and the bacteriostatic activity of macrophages against Mycobacterium tuberculosis (35). IL-4 may mediate proliferative effects in macrophages through the modulation of L-arginine metabolism, induction of MHC class II expression, and stimulation of mannose receptor endocytic activity (33, 36), which recently has been reported to enhance phagocytosis against F. tularensis (37). Mast cell-derived IL-4 may control intramacrophage LVS growth by inducing a stimulatory effect on macrophages to undergo cellular proliferation and prevent the induction of apoptosis (38). A reduction in Caspase 3-mediated apoptosis in LVS-infected macrophages on coculture with mast cells (A.R.R. and B.P.A., unpublished observations) has been documented. Importantly, we have observed an in vivo relevance to these findings and found greater bacterial burden and heightened susceptibility in mast cell-deficient and IL-4R⫺/⫺ mice. Reconstitution of the mast cell compartment restored the protective Ketavarapu et al.
ability against pulmonary LVS infection in W/Wv mice, suggesting that the greater susceptibility of these animals was because of the deficiency of mast cells, but not other abnormalities. Additionally, mast cell-deficient mice displayed greater inability to control the bacterial infection than IL-4R⫺/⫺ mice, suggesting that mast cells may use IL-4-dependent and -independent mechanisms in vivo. However, adoptive transfer of IL-4-deficient or -competent mast cells into mast cell-deficient animals reconstituted comparable levels of protection suggesting a reduced role of mast cell-derived IL-4 in vivo. This latter observation may result from: (i) the presence of alternative cellular sources of IL-4 as evidenced by IL-4 expression within the lymph nodes of mast cell-deficient animals, (ii) the masking of the effect of IL-4 by the significant contribution of contact-dependent events and other secreted products of mast cells. To this end, stabilization of mast cells, despite initial greater body weight loss and lung bacterial burden, did not affect survival after pulmonary LVS challenge, suggesting the considerable contribution of contact-dependent events. TNF-␣ and IFN-␥ have been reported to be critical for early control of LVS intramacrophage growth (8, 9, 39–41). However, the results from this study provide a different dimension to the conventional views of innate defenses against Francisella, including the role of mast cells and IL-4 in controlling intramacrophage Francisella replication. This alternative pathway may shape the innate immune response and subsequent host defenses against pulmonary Francisella infection. Materials and Methods Bacteria. F. tularensis LVS (lot 703-0303-016 from R. Lyons at University of New Mexico) was grown in trypticase soy broth (TSB) supplemented with 0.1% cysteine. Mice. Specific pathogen-free mice (5– 8 weeks) were used in all experiments. C57BL/6 IL-4⫺/⫺ (42) and BALB/c IL-4R⫺/⫺ (43) and matching wild-type mice were purchased from Jackson Laboratories. Mast cell-deficient WBB6F1-KitW/ KitW-v mice (44) that have mutations in both copies of c-kit and congenic wild-type WBB6F1⫹/⫹ mice were obtained from Jackson Laboratories. Mice were bred and homozygous WBB6F1⫹/⫹ (WT) and WBB6F1W/Wv (W/Wv) animals were used in experiments. BALB/c STAT6⫺/⫺ mice (45) were obtained PNAS 兩 July 8, 2008 兩 vol. 105 兩 no. 27 兩 9317
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Fig. 4. The contribution of mast cells in innate defenses against pulmonary LVS challenge. Four- to 6-week-old WT or W/Wv mice (six animals per group) challenged i.n. with LVS (3,000 cfu). (A) Staining for mast cells (pink) in the lymph node sections of WT and W/Wv mice. (B) Survival analysis. P ⬍ 0.05 (Kaplan–Meier test) compared with WT mice. (C) Bacteria recovered from the lungs, liver, or spleen of W/Wv or WT mice (four animals per group) on day 3 or 6 after i.n. LVS challenge. *, P ⬍ 0.001 (two-way ANOVA) compared with W/Wv mice. (D) Sections were stained for mast cells (anti FcRI␣, green), or IL-4 (anti IL-4, red). Overlay (yellow) is indicative of colocalization of mast cells with IL-4. Results are representative of two to three independent experiments.
from the University of Texas Health Science Center, San Antonio. Detailed information is listed in SI Methods. Histology and Immunofluorescence Staining. Lymph nodes were removed from mice at 0, 3, or 5 days postchallenge, fixed, and stained with hematoxylin and eosin (H&E), or with naphthol AS-D chloroacetate esterase (Sigma) to visualize mast cells (25). In some experiments, tissue sections were stained with fluorescein isothiocyanate (FITC) conjugated anti-mouse FcRI (e-Bioscience) followed by R-phycoerythrin (PE)-conjugated rat anti-mouse IL-4 (BD Biosciences) and Hoechst nuclear stain (Sigma). Detailed information is listed in SI Methods.
When transwell plates were used, mast cells were seeded into the top wells and macrophages into bottom wells. The cells were infected with F. tularensis LVS at a MOI of 10 or 100, and bacterial enumeration and cytokine analyses were performed at indicated time points. Detailed information is listed in SI Methods. Statistical Analysis. Survival data were analyzed by the Kaplan–Meier test and bacterial counts and cytokine analyses were evaluated by Student’s t test or ANOVA by using the statistical software program SigmaStat.
Generation of Primary Cells and in Vitro Infections. Primary mast cells and macrophages were prepared from bone marrow cells as described in SI Methods. Primary cells were seeded individually or as cocultures into culture plates.
ACKNOWLEDGMENTS. This work was supported by National Institutes of Health Grant PO1 AI057986. We thank Dr. Peter Dube and Leslie Linehan from University of Texas Health Science Center, San Antonio, and Heather Powell from University of Texas, San Antonio, for technical expertise, and also Dr. Dale Selby (Wilford Hall Medical Center, San Antonio) for histopathological evaluation.
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