As an understanding of the potential health risks associated with the fabrication and use of engineered nanomaterials, including SWCNT, emerges, there is an increasing need for their detailed toxicological assessment. Realistic exposures to SWCNT are likely to occur in conjunction with other pathogenic impacts, such as microbial infections. Since both SWCNT and infections induce inflammatory response and oxidative stress, their interactions may trigger interactive reactions that are difficult to assess by simple additive extrapolations. Here, we report that sequential exposure to SWCNT and LM triggered an enhanced inflammatory response. The amounts of inflammatory cells in BAL, collagen deposition, and cytokine responses were all markedly enhanced compared with the respective responses to each of the components alone.
We previously demonstrated that toxic responses to SWCNT could be modulated by a number of factors such as genetic and nutritional status (21
) (e.g., vitamin E levels, expression of NADPH oxidase). Thus, individual susceptibility to toxic effects of SWCNT may vary. Here, we documented that bacterial infection can act as an additional important factor to enhance inflammatory response to SWCNT adding yet another aspect of modified sensitivity to SWCNT toxicity. Different modes of exposure to SWCNT and LM—simultaneous or sequential—may result in different patterns of deposition and clearance. In this study, we were mostly interested in the interaction between SWCNT-induced inflammatory response and infectivity. Therefore, we chose the regimen of pre-exposure to SWCNT associated with pronounced inflammatory and oxidative stress responses. Moreover, our previous studies (13
) have demonstrated that after 3 days of exposure to SWCNT, a significant part of nanotubes in the lung is sequestered in granulomas thus limiting direct physical contact and potential adsorption of LM on SWCNT.
The importance of enhanced responses to a sequential exposure to SWCNT and LM dictates the necessity to better understand the pathways involved in their interactions. Recently, attempts have been made to use nanoparticles for targeted drug delivery to specific cells of the immune system. Macrophages may be very promising targets for drug-loaded nanoparticles due to their central role in inflammation and their ability to harbor a variety of bacteria, viruses, and parasites. This sequestering of infectious agents is significant for risk of contracting a number of deadly diseases in humans, such as leishmaniasis, tuberculosis, and HIV (22
). Special consideration, however, should be given to the inflammatory potential of drug-laden nanoparticles with SWCNT-like properties, since SWCNT may modulate the sensitivity of the body's immune responses to infections. The current findings indicate that SWCNT exacerbate pro-inflammatory and pro-oxidant injuries or effects. In addition, synergies in induction and release of specific cytokines may be an important participating mechanism underlying the risks of unexpected side effects of co-exposures of the SWCNT-like nanoparticles with bacteria or other environmental insults.
While engineered nonmodified SWCNT used in the study may compromise innate immune response and clearance of bacterial pathogens, thus raising questions regarding possible health aspects for producers and users (24
), there are many examples of successful use of nanomaterials for delivery of anti-inflammatory agents such as antibiotics as well as steroidal (25
) and nonsteroidal anti-inflammatory drugs (NSAIDs) (26
). In addition, immunonanoparticles (i.e., nanoparticles functionalized with pathogen-specific antibodies) may serve as antimicrobial carriers for improving the stability and activity of antimicrobials in foods. A recent study documented that the use of polystyrene immunonanoparticles with active carboxyl groups conjugated with anti-LM antibody and coated with lysozyme was more effective than direct addition of lysozyme for inactivating L. monocytogenes
). Moreover, some nanoparticles (e.g., cerium oxide [CeO2
]) exert significant anti-inflammatory and antioxidant properties (28
). Further, different types of nanoparticles (e.g., silver nanoparticles, W(4+)-doped titanium-coated nickel ferrite composite nanoparticles) without any specific loads reportedly exert antimicrobial activity by themselves or in combination with photo-therapy (29
). Finally, antimicrobial effects of nanoparticles seem to be dependent on the type of bacterial pathogen. A recent report, documented that pristine SWCNT can exhibit strong antimicrobial activity toward Escherichia coli
). The authors speculate that direct cell contact with SWCNT can cause severe membrane damage and subsequent cell inactivation. Obviously, further research on interactions of different types of nonmodified, purposely modified nanoparticles and nanoparticles loaded with bioactive cargoes with different bacterial pathogens is warranted.
The mechanism by which pre-exposure to SWCNT decreases subsequent bacterial clearance is unclear. Our experiments demonstrate that impaired innate clearance of LM in mice pre-exposed to SWCNT may be due, at least in part, to a decreased potency of macrophages in handling this bacterial pathogen. This was evidenced by a decreased capacity of alveolar macrophages obtained from mice pre-treated with SWCNT to phagocytize LM. Interestingly, even pre-incubation of “normal” AMs isolated from naïve mice to SWCNT affected their phagocytozing function and decreased uptake of LM. Specific mechanisms underlying the effects of SWCNT on macrophage functions remain to be elucidated. Interestingly, no effects of SWCNT on superoxide production by AMs were detected (data not shown). The ability of alveolar macrophages to generate NO, however, was significantly compromised after SWCNT pre-exposures of mice in vivo or pre-incubation with macrophages in vitro. This suggests that NO-dependent (rather than superoxide-dependent) pathways affected by SWCNT in macrophages might be involved in decreased clearance of LM in SWCNT pre-exposed mice.
It is possible that killing of LM depends more on production of nitric oxide (NO) than ROS from the oxidative burst, since genetic deletion of iNOS produces a more severe decrement in resistance to LM than deficiency of p47phox, a major component of the 47-kD phagocyte NADPH oxidase (34
). On the other hand, Saxena and coworkers showed that exposure of mouse AMs to diesel exhaust particles attenuated IFN-γ–induced production of NO (36
). In addition, pulmonary exposure of rats to diesel exhaust particles depressed NO production by AMs in response to pulmonary LM infection and depressed bacterial clearance (37
). Similarly, pulmonary exposure to ROFA enhanced zymosan-stimulated chemiluminescence in rat AMs but reduced NO release and bacterial clearance in response to the same stimulus (38
Host resistance to the intracellular bacterium, LM, involves both innate and adaptive immune mechanisms (39
). This organism has been used to demonstrate the ability of other particulate toxins such as diesel exhaust particles (37
), residual oil fly ash (38
), and welding fumes (40
) to inhibit host defense mechanisms and increase the severity of pulmonary infections. Initial innate immune responses to infection include accumulation and activation of neutrophils, natural killer (NK) cells, and macrophages to sites of infection with attendant up-regulation of bacteriocidal activities. If organism load is large or infection persists, then secondary Th1-type dominant adaptive immune strategies are required for protection. Evidence for this secondary response includes a requirement for induced TNF-α (41
) and IFN-γ (42
) expression to mediate resistance to LM.
Our data demonstrate that pre-exposure to SWCNT greatly attenuated the clearance of LM from the lung. Using larger doses of LM in a rat model, Yin and colleagues attributed the immunosuppressive effects of acute exposure to diesel exhaust particles to attenuation of early macrophage activation and cytokine release (43
). The cytokine profiles obtained in our study 3 days after infection (Experimental Day 6) appeared to be very similar between uninfected animals pre-treated with PBS or both doses of SWCNT. We could not readily detect TNF-α or IL-1β at the early 3-day time point of experiment, but robust elevations in IL-6, G-CSF, MCP-1, and other chemokines were observed in all cases. This cytokine profile might reflect a less robust Th1 response arising from SWCNT treatment. However, this decrease was similar with both doses of SWCNT, whereas only high-dose SWCNT showed significant compromise of host defense. In addition, IL-12p40 subunit was equivalently induced in all groups. Thus, it appears that the overall ability of LM to initiate early cytokine responses after infection is not greatly affected by SWCNT exposure. This suggests that the initial innate signaling mechanisms for pathogen recognition and responses remain intact. In contrast, it is possible that target cell responses to these cytokines are decreased by SWCNT.
The failure to clear LM early after infection in mice pre-exposed to SWCNT leads to a continued elevation in nearly all the chemokines and acute phase cytokines into the later course of infection. In fact many of these, such as KC, CCL3, CCL4, and CCL5, are significantly higher in the high-dose SWCNT mice on Day 7 after infection (Experimental Day 10) compared with the levels seen in the same group 3 days after infection (Experimental Day 6), despite the fact that organism load appears similar. A significant induction of a number of cytokines/chemokines on Experimental Day 6 coincided with the increase in cell recruitment at this time point, which, however, was not different for ± SWCNT experimental groups. Importantly, assessments of the cells already present in the lung probably reflect cytokine/chemokine production at some point earlier. It is possible that differential cytokine production occurred earlier, for example on Days 3 to 5. For example, it is clear that the exposure to SWCNT alone for 3 days was accompanied by a robust induction of chemokines and other inflammatory mediators (). Thus, it is likely that SWCNT-dependent influx of some inflammatory cells had already occurred in the lung at the time of LM infection. The reasons for the synergistic effects on cytokine release are unclear, but could reflect the shift from innate immune mechanisms to adaptive Th1 lymphocyte–dominant immune-dependent responses. The accumulation of lymphocytes antecedes the accumulation of other inflammatory cells, and was most obvious in the SWCNT-treated groups at the latest time point. Moreover, the levels of IL-17 and IFN-γ in BAL paralleled this pattern and were significantly elevated after high-dose SWCNT compared with LM-infected animals pre-treated with PBS. Both of these lymphokines are known to potentiate release of inflammatory mediators from target cells on combination with other inducing stimuli (44
). We observed an early induction of IL-12p70, a primary determinant of Th1-biased response, in all the LM-infected groups. Only mice pre-exposed to high-dose SWCNT, however, showed a sequential release of the Th1 effector product, IFN-γ. Therefore, IL-12p70 alone is not sufficient for the maximal appearance of IFN-γ. In addition, other sources of IFN-γ, such as NK and γδ-T cells, also need to be considered as potential sources of IFN-γ during mixed exposures to LM and SWCNT. Our data also indicate that the newly described Th17-dependent immune response (46
) is activated during LM infection in the lung, especially during SWCNT exposure.
Previous work from our group has established that SWCNT exposure promotes tissue fibrosis and granuloma formation associated with the release of high levels of TGF-β (19
). While TGF-β is well known for its ability to direct tissue remodeling and repair, it also possesses immunoregulatory properties. TGF-β produced by regulatory T cells (Tregs) has immunosuppressant properties by suppressing Th1 effector cell responses (47
) and could, thus, limit adaptive immune responses to LM in the SWCNT-exposed lung. Moreover, in the presence of IL-6, it functions to drive Th17 differentiation (48
) and may account for the elevated levels of IL-17 observed in infected animals exposed to a high dose of SWCNT. Thus, it is possible that SWCNT-dependent chronic inflammation/fibrosis can modulate the magnitude and type of adaptive immune responses generated upon challenge of that organ to a microbial pathogen. While not specifically measured in this study, we have shown that TGF-β peaks 3 days after SWCNT exposure (i.e., the same time as when we inoculated the lungs with LM) (13
). It is also evident that the lung environment produced by SWCNT alone contained high levels of other immunomodulatory mediators, such as IL-12 family members, G-CSF, IL-6, IL-5, MIP-1α, and MCP-1, at the time of LM infection (i.e., Day 3 of experiment) (), that may also modulate the repertoire of host-defense responses to microbial pathogens.
Th2 immune responses and IL12-p40 are associated with fibrotic tissue remodeling (49
), while Th1 responses are a requisite for efficient clearance of facultative intracellular pathogens like Listeria
. We can speculate that the Th2/Th1 bias of immune response is altered during SWCNT/LM co-exposure. The burst of IFN-γ in co-exposed animals may reflect aspects of Th1-based immunity; however, the SWCNT-associated component of inflammation may have modified the immune response to favor the development of pro-fibrogenic environment, as illustrated by declining levels of IL-12p70 and augmentation of IL-12p40. In addition, SWCNT exposure alone produced alterations in several cytokines, like IL-5, MCP-1, IL-6, and G-CSF, previously associated with Th2 responses (51
). The fact the IL-12p40 homodimers can antagonize the biologic effects of IL-12p70 may also be of relevance here (54
), since we frequently observed induction of IL-12p40 in absence of any changes in mature IL-12p70.
In line with this, we found a markedly increased level of collagen deposition in the lung of mice exposed to SWCNT/LM. These results were confirmed by morphometric assessments of alveolar wall thickening (data not shown). While the collagen measurements were performed on Day 10 of the experiment, which may be viewed as an early time point to characterize fibrotic development in the lung, our previous studies have established that the early SWCNT-induced collagen deposition did not resolve and progressed over at least 60 days (13
). Thus it is likely that LM enhances SWCNT-induced early fibrotic response.
SWCNT doses used in the study are occupationally (rather than environmentally) relevant. Previously we have reported that a 20-μg dose of SWCNT given by pharyngeal aspiration route to C57BL/6 mice deposited in the alveolar region, corresponding to approximately the same estimated dose for a worker exposed to the OSHA PEL for graphite (5 mg/m3
, 8 h workday, 40 h per week) over a period of 20 work days (13
). According to our earlier published data on the airborne SWCNT concentrations in a laboratory producing SWCNT, peak airborne concentrations of 53 mg/m3
were measured. Aspirated doses of 10 and 40 μg SWCNT/mouse are relevant to predicted deposited doses after 2 and 8 years of exposure at peak airborne concentrations measured in these occupational settings (55
In summary, our findings clearly demonstrate that sequential exposure to SWCNT and LM induced unusual responses in which both components—nanoparticles and bacterial infection—mutually enhanced inflammation and depress bacterial clearance.