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Previously, treatment with anti-gpTNFα antibody enhanced TNFα mRNA expression in pulmonary granulomas microdissected from non-vaccinated guinea pigs, and modified splenic granuloma architecture. In this study, pleural fluid, cells, and granulomatous tissues were collected 3, 5, and 8 days post-pleurisy induction in guinea pigs treated with anti-gpTNFα or normal serum control. Neutralizing TNFα reduced the percentage of macrophages in the pleural exudate while increasing the proportions of neutrophils and lymphocytes. Cell associated mycobacterial loads were increased in guinea pigs treated with anti-gpTNFα antibody. Cells from the pleural exudate in both treatment groups at day 3 expressed predominantly TNFα and IFNγ mRNA. By day 5, treatment with anti-gpTNFα antibody significantly reduced TNFα mRNA and increased TGFβ and iNOS mRNA expression, a transition which did not occur in the control group until day 8. TNFα mRNA overwhelmed the cytokine milieu of microdissected pleural granulomas in the control group at day 3 whereas TNFα, IFNγ, and TGFβ mRNA dominated the anti-gpTNFα-treated group. At day 8, granulomas from the control group began shifting towards an anti-inflammatory profile with increased levels of TGFβ mRNA. Neutralization of TNFα hastened the transition to an anti-inflammatory cytokine response in guinea pig pleural granulomas and exudate cells.
One of the key outcomes of in vivo mycobacterial infection is the formation of granulomas. This structure is maintained and stabilized by events mediated by both host and pathogen. It is believed that bacteria can live for prolonged periods of time within the environment of a granuloma while, at the same time, bacterial spreading to other areas of the organism is restricted . In this sense, granulomas represent a fine example of the delicate balance that keeps the infection in equilibrium-an equilibrium that, in most otherwise healthy individuals, does not harm the host but that does not kill the bacterium either. These events lead to the secretion of pro-inflammatory cytokines and chemokines, including TNFα . On the other hand, TNFα also has been implicated in the pathologic response of the host to M. tuberculosis infection and is often cited as a major factor in host-mediated destruction of lung tissue. Tipping the balance of TNFα in the lungs may lead to increased pathology and necrosis.
The importance of TNFα in the control of Mycobacterium tuberculosis (M. tb) is due to its role as a mediator of macrophage activation. A number of groups have shown that granuloma formation in tuberculosis in the absence of TNFα is disorganized, with fewer activated or epithelioid macrophages . Clearly, TNFα affects cell migration and influences expression of adhesion molecules as well as chemokines and chemokine receptors, and this is certain to affect formation of functional granulomas in infected tissues. Our laboratory has shown that guinea pig macrophages infected with M. tb release inflammatory cytokines such as interleukin-1 (IL-1), IL-6, and TNFα [3–5]. Recently, we have demonstrated that resident peritoneal macrophages from BCG-vaccinated guinea pigs stimulated in vitro with recombinant guinea pig (rgp) TNFα and/or rgp IFNγ exhibited a significant increase in of H2O2 production, MHC class II expression, and IL-12p40 mRNA production . Neutralizing endogenous TNFα in cocultures of T-cells and macrophages from BCG-vaccinated guinea pigs down-regulated the expression of IL-12p40 and IFNγ mRNA while increasing intracellular bacterial growth . In vivo neutralization of TNFα in BCG-vaccinated guinea pigs for 3 weeks post aerosol infection with virulent M. tb resulted in significant splenomegaly. Furthermore, granulomas microdissected from non-vaccinated guinea pigs infected with M. tb were overwhelmed with TNFα mRNA at 3 and 6 weeks post-infection compared to those from BCG-vaccinated guinea pigs in which Type 1 cytokine mRNA (IFNγ, IL-12p40) at 3 weeks post-infection are replacd by TGFβ mRNA at 6 weeks . Similarly, lung macrophages from BCG-vaccinated guinea pigs showed increased TNFα mRNA expression in response to antigen-specific and mitogen stimulation . The multiple mechanisms by which TNFα promotes effective granuloma formation, maintenance, and function have yet to be determined, especially in the highly relevant guinea pig model.
The guinea pig model of pleurisy is remarkably similar to that seen in humans as a rapid inflammatory response occurs in the pleural space of animals sensitized by BCG vaccination and injected intra-pleurally with either killed or viable M.tb. We have shown that the pleural inflammatory response can be manipulated by treatment with recombinant cytokines or neutralizing antibody to cytokines to provide insight into the local immunoregulatory mechanisms [10–13]. Neutralization of endogenous TGFβ throughout the course of disease in guinea pigs induced with pleurisy resulted in a significant reduction in the percentage of lymphocytes and production of IFNγ and CCL5 mRNA in the pleural exudates, while the percentage of neutrophils and CXCL8 mRNA expression were upregulated, indicating that modulation of cytokine function was feasible with this model . In this study, we set out to determine the effect of TNFα neutralization on the inflammatory response and granuloma formation in pleurisy-induced guinea pigs. This model was chosen as it would allow us to inject neutralizing antibody directly into the infected cavity and affect granuloma formation throughout the course of the study. In addition, the focused effect of the neutralizing antibody within a defined anatomical site was expected to mitigate against a dilution of the antibody throughout the entire body.
Specific, pathogen-free outbred Hartley strain guinea pigs (250–300 g) from Charles River Breeding Laboratories, Inc. (Willmington, MA) were housed individually and provided commercial chow and tap water ad libitum. They were maintained in a temperature- and humidity-controlled environment and exposed to a 12-h light/dark cycle. They were vaccinated intradermally (103 CFU) with Mycobacterium bovis BCG (Danish 1331 strain; Statens Seruminstitut) and allowed to rest for 6 weeks before pleuritis induction. Following virulent infection, the guinea pigs were housed under ABSL-3 containment. All protocols were approved by the Texas A&M University Laboratory Animal Care Committee.
New Zealand white rabbits (2–3 kg) were immunized with recombinant guinea pig (rgp) TNFα by the method previously published by our laboratory . The rgp-TNFα protein was produced according to our established method [4, 14]. The serum collected at the end of the immunization protocol was tested by Western blot for reactivity against rgpTNFα as previously described . A dilution of 1:5000 was able to detect as little as 50 ng of rgpTNFα (data not shown). Anti-gpTNFα produced under these conditions has been shown to neutralize gpTNFα in an L929 cell bioassay .
M. tuberculosis H37Rv (ATCC 27294) was maintained in stock suspensions of known viability and stored at −80°C. BCG vaccinated guinea pigs were anesthetized by intramuscular injection of ketamine (30 mg/kg) and xylazine (2.5 mg/kg), and one milliliter of 2 × 108 CFU/ml bacteria was injected bilaterally into the pleural space using our previously published method [11, 15]. The guinea pigs were anesthetized and given daily intrapleural injections of either undiluted rabbit anti-gpTNFα antibody or normal rabbit serum (1 milliliter per guinea pig, per day) beginning on the day of infection. Groups of animals were sacrificed on days 3, 5, and 8 post-induction of pleuritis.
Guinea pigs were euthanized by intraperitoneal injection (100 mg/kg) of sodium pentobarbital (Sleepaway; Fort Dodge Laboratories, Inc., Fort Dodge, IA). Using our previously published method , the abdominal cavity was opened, a small incision was made at the top of the diaphragm and the accumulated fluid and cells were removed. Samples were spun at 1200 x g for 15 minutes at room temperature. Cell viability was determined by Trypan Blue exclusion. The cell pellets and cell-free supernatants were collected for intracellular and extracellular bacteria analyses, respectively. Samples were plated onto 7H10 agar plates and incubated at 37°C. Colony forming unit (CFU) counts were recorded after 21 days.
Fluid retrieved from the pleural cavity was centrifuged onto silanated glass slides (CSA-100; PGC Scientifics, Gaithersburg, MD) at 140 x g for 5 min using a Cytospin 2 (Shandon Southern Instrument, Inc., Sewickley, PA). Cells were stained with Diff-Quik (Dade Behring Inc., Newark, Del) and viewed under a microscope to determine the relative cell composition based on morphology.
The diaphragm and surrounding connective tissues containing visible granulomas were excised aseptically. Each tissue was then placed into 10% buffered-formalin for 1 h at 4°C, and transferred into 70% EtOH. Tissue processing and LCM was performed as previously described [8, 13].
Total RNA was isolated from pleural effusion cells and microdissected granulomas as previously published [8, 13]. Reverse transcription and real-time PCR, performed by using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), were carried out as previously described using the primer sequences for the guinea pig IL-12p40, TNFα, TGFβ1, IFNγ, IL-10, iNOS, and HPRT published previously [6, 11, 13, 14]. Data obtained from pleural exudates were normalized to HPRT mRNA expression of the same sample. Expression values from microdissected granulomas were normalized to HPRT mRNA expression and then to nongranulomatous tissues obtained from the same animal.
A two-way analysis of variance (ANOVA) was used to examine statistical differences between treatment groups (normal vs. anti-gpTNFα antibody) at the 95% confidence interval.
Grossly visible granulomas were not detected in the diaphragm or surrounding tissues in pleurisy-induced guinea pigs until day 8. Granulomas from the control serum group were large, with an intensely necrotic center surrounded by loosely grouped scatterings of epithelioid macrophages and lymphocytes (data not shown). While the cellular concentration in the effusion was not affected by anti-TNFα treatment, the cellular composition was (Table 1). The absence of necrosis in the pleural granulomas of anti-gpTNFα antibody-treated guinea pigs is consistent with the significant reduction in the proportion of macrophages in the effusions. No necrosis was detected in any of the granulomas in antibody treated guinea pigs, suggesting that TNFα may play a central role in the development of necrosis in this disease model.
Fluid was collected from the pleural space of guinea pigs infected with M. tb H37Rv at 3, 5, and 8 days post-treatment. The cellular concentration of the accumulated fluid increased gradually over the experimental time course and was similar in guinea pigs treated with either anti-gpTNFα antibody or control serum (Fig. 1A). Cell-associated bacillary loads from both treatment groups remained constant between days 3 & 5 and declined markedly by day 8 (Fig. 1B). Cell-associated bacterial loads in the pleural effusions of guinea pigs treated with the anti-gpTNFα antibody were consistently lower than in the serum control group, although statistical significance was achieved only on day 8 (Fig. 1B). Extracellular bacilli from the same samples were markedly lower compared to cell-associated mycobacteria, and the levels were similar in both treatment groups throughout the experiment (Fig. 1C). The pleural fluid from guinea pigs treated with the control serum was comprised mostly of neutrophils (40–45%), macrophages (~35%), and lymphocytes (20–25%) (Table 1). Treatment with anti-gpTNFα significantly reduced the presence of macrophages while significantly increasing the proportions of neutrophils and lymphocytes. This trend was seen throughout the course of the 8-day study.
Analysis of cytokine mRNA expression of the pleural effusion cells revealed predominant expression of TNFα mRNA and, to a much lesser extent, IFNγ mRNA in both treatment groups at 3 days post-treatment (Fig. 2). The presence of TGFβ, IL-12 p40, IL-10, and iNOS mRNA were minimal in both animal groups. Interestingly, TNFα mRNA expression levels were markedly higher (30%) in the group treated with anti-gpTNFα antibody at 3 days post-treatment compared to the control serum group (Fig. 2). However, IFNγ mRNA expression was reduced by 1.5-fold in this same group. By day 5 post-treatment, all cytokine mRNA expression levels in the pleural effusion cells had increased (Fig. 2). The expression of TNFα mRNA continued to prevail over the other cytokines measured in the guinea pigs injected with the control serum. In general, relative expression levels of TGFβ and iNOS mRNA and, to a lesser extent, IL-12p40 and IL-10 mRNA increased substantially compared to day 3 post-treatment in the same treatment group. Treatment with anti-gpTNFα antibody at day 5 attenuated TNFα mRNA expression by 3-fold while increasing TGFβ, IL-12p40, IL-10, and iNOS mRNA expression levels by 2-6 fold (Fig. 2). Antibody treatment did not affect IFNγ mRNA production, however. At the 8-day interval, the mRNA expression levels of TNFα in the animals treated with anti-gpTNF antibody had subsided dramatically to levels comparable to the control serum group (Fig. 2). IFNγ and IL-10 mRNA expression levels remained low and were not affected by antibody treatment at 8 days post-treatment. However, TGFβ, IL-12p40, and iNOS mRNA expression levels remained elevated (2–9-fold) in guinea pigs treated with the anti-gpTNFα antibody when compared to the control serum group (Fig. 2).
Comprehensive charts were constructed to illustrate the relative proportions of mRNA transcripts for each of the six cytokines measured in the pleural fluid throughout the course of the study (Fig. 3). These charts clearly depict the dominant presence of the pro-inflammatory cytokine, TNFα mRNA and, to a lesser extent, IFNγ mRNA at day 3 post-treatment in both animal treatment groups. At day 5 post-treatment, cells from the control group continued to produce abundant amounts of TNFα mRNA and it was at this time point when a switch from pro- to anti-inflammatory cytokine mRNA production was seen. TGFβ and iNOS mRNA expression levels were approximately ¼ of the mRNA pool at day 5, and then increased dramatically by day 8 (Fig. 3). At day 5, the mRNA cytokine composition of the anti-gpTNFα antibody treatment group was predominantly comprised of TGFβ and iNOS mRNA, which is strikingly similar to the profile seen by the control serum group at day 8 post-treatment.
To determine whether TNFα neutralization affected cytokine mRNA production in the developing granulomas of the pleurisy model, the granulomas from the diaphragm and connective tissues were microdissected by laser capture microdissection (LCM). Granulomas from the control serum group expressed high levels of TNFα mRNA, followed by IFNγ, TGFβ, and IL-12p40 mRNA (Fig. 4) up to day 5 post-treatment. Treatment with anti-gpTNFα antibody reduced TNFα and IFNγ mRNA expression levels even further (~700-fold), while significantly increasing IFNγ, TGFβ, IL-12p40, and iNOS mRNA expression levels (2–20-fold) (Fig. 4).
The relative proportions of mRNA transcripts are displayed in Figure 5. Granulomas microdissected from the control serum group were overwhelmed by TNFα mRNA up to day 5 post-treatment. By day 8, the presence of TGFβ mRNA became apparent. TNFα neutralization altered cytokine mRNA expression levels in granulomas as early as 3 days post-treatment (Fig. 5). At day 3, the granulomas from the anti-gpTNFα antibody-treated guinea pigs were predominantly composed of cells producing TNFα mRNA and to a lesser extent, TGFβ and IFNγ mRNA. By day 5, IFNγ mRNA production began to take over the granulomas with a concomitant reduction in TNFα mRNA expression. At day 8 post-treatment, TGFβ mRNA production dominated and the presence of IFNγ and TNFα mRNA was greatly attenuated.
We have previously demonstrated that splenocytes from previously vaccinated guinea pigs stimulated with antigen and rgpTNFα resulted in enhanced lymphoproliferation and IFNγ mRNA production . Also, alveolar and peritoneal macrophages from non-vaccinated animals stimulated in vitro with M. tb and rgpTNFα upregulated TNFα and IL-12p40 mRNA expression levels . These same cell cultures treated with antibody to neutralize TNFα increased intracellular bacillary growth. Furthermore, neutralizing TNFα antibody decreased the mRNA production of the pro-inflammatory IL-1β cytokine in cocultures containing alveolar macrophages and neutrophils infected with virulent M. tb . It is clear that tipping the balance of TNFα during the immunological response to M. tb infection in guinea pigs alters the cytokine milieu and the intracellular fate of virulent mycobacteria.
Our laboratory previously observed that the cytokine milieu of pulmonary granulomas from non-vaccinated guinea pigs following aerosol infection with M. tb was overwhelmed by TNFα mRNA . Moreover, cytokine mRNA analysis of splenic granulomas from non-vaccinated animals showed close resemblance to primary granulomas . These data reflect the importance of TNFα in granuloma formation and maintenance. In the current study, we observed that TNFα neutralization decreased cellularity and necrosis of granulomas in the diaphragm of guinea pigs (data not shown). Similarly, Chakravarty, et al recently demonstrated that TNFα neutralization induced dissolution of lymphoid aggregates in the lungs of mice infected with M.tb . We have previously observed that in vivo neutralization of TNFα in BCG-vaccinated guinea pigs aerosol-infected with M.tb resulted in significant splenomegaly . Taken together these results clearly show a role for TNFα in the immunopathological response of tuberculosis, specifically in the aggregation of lymphocytes and the development of a necrotic center in the granuloma.
We observed the effect of anti-gpTNFα antibody treatment on TNFα and TGFβ mRNA expression in the pleural exudates as early as day 3 post-treatment (Fig. 2). Interestingly, TNFα mRNA expression levels were enhanced at this time point which suggests a cellular compensatory mechanism to overcome the drop in TNFα protein by increasing gene transcription. It is known that TNFα shares many similar properties with IL-1β and, therefore, it is possible that the removal of TNFα protein resulted in the upregulation of IL-1β protein which, in turn, can stimulate TNFα production in an autocrine manner . Accordingly, a recent study in our laboratory using neutralizing anti-gpTNFα antibody resulted in a transient increase in TNFα mRNA in cocultures infected in vitro with live M.tb . Interestingly, treatment with anti-gpTNFα antibody decreased IFNγ mRNA expression in the pleural exudates at day 3, which implies a TNF-dependent mechanism (Fig. 2). TNFα and IFNγ are pleiotrophic cytokines that have been shown to function either cooperatively or antagonistically . It has also been demonstrated that TNFα may directly or indirectly regulate the expression of IFNγ and vice versa in many in vitro models . By day 5 & 8 post-treatment, TGFβ, IL-12p40 and iNOS were enhanced by TNFα neutralization demonstrating an anti-inflammatory profile taking shape.
Treatment with anti-gpTNFα antibody had a much more rapid effect on granuloma cytokine mRNA profiles than on the response of pleural effusion cells (Fig. 3). As early as 3 days post-treatment, the cytokine mRNA milieu of granulomas from the control serum and anti-gpTNFα antibody treated guinea pigs was significantly different. The results from this study clearly indicate that TNFα neutralization hastens the resolution of the pleuritis response as the anti-inflammmatory TGFβ mRNA expression was upregulated at earlier time points in both the pleural exudates and the tissue granulomas from anti-gpTNFα treated guinea pigs (Fig. 3 & 5). A recent study by Chakravarty, et al (2008) demonstrated that neutralization of TNFα in the mouse model of tuberculosis results in disorganization of the tuberculous granuloma and enhanced expression of various proinflammatory cytokines. The discrepancy between their results and ours can be explained by use of different animal models (mouse vs. guinea pig) and different routes of infection (aerosol vs. intrapleural). Our results also contradict previous results from our laboratory which demonstrated that systemic treatment with anti-gpTNFα in guinea pigs aerosol infected with M. tb for 3 weeks resulted in significant splenomegaly and disorganization of granulomas in the spleen . Again, this can be explained, in part, by the different infection routes chosen.
TNFα is produced mainly by macrophages and daily treatment with anti-gpTNFα antibody resulted in a decrease in macrophage populations, and an increase in neutrophil and lymphocyte populations throughout the course of the study (Table 1). Although the absolute number of cells between animal groups did not differ (Fig. 1A), the cellular composition and cytokine mRNA profile by day 5 & 8 post-treatment were markedly different. The rapid increase in lymphocyte and neutrophil populations and the elevated TGFβ and iNOS mRNA expression counteracted the proinflammatory effects of TNFα and IFNγ cytokine mRNA seen at day 3 (Table 1 & Fig. 2). As mentioned earlier, this resulted in a reduction in cellularity and the appearance of necrosis (data not shown). During the early phase of antibody treatment (day 3 & 5), the existing macrophages may have responded to the loss of TNFα protein with the overproduction of TNFα mRNA. However, by day 8 post-treatment, the relative loss of macrophages accompanied by increasing populations of neutrophils and lymphocytes helped shift the cytokine milieu to an anti-inflammatory profile in anti-gpTNFα treated guinea pigs.
Induction of pleurisy in the guinea pig requires prior vaccination to develop a rapid local immune response. Cell-associated bacillary loads were significantly reduced by day 8 post-treatment in both animal groups (Fig. 1B), indicating successful vaccination. These data are in contrast with our previous results whereby pulmonary granulomas microdissected from BCG-vaccinated guinea pigs were overwhelmed with TGFβ mRNA transcripts at 6 weeks post-infection . TGFβ is known to inhibit several events in T-cell and macrophage activation resulting in low production and/or function of antimicrobial mediators pertinent to the host immune response . The discrepancy in these results can be explained, in part, by the route and dose of infection, and the timing of the study.
Neutralizing TNFα decreased cell-associated mycobacterial loads in the pleural exudates while not affecting the levels of extracellular bacilli throughout the course of the study (Fig. 1B & 1C). However, TNFα mRNA expression levels in the pleural effusion cells were increased at day 3 post-treatment and decreased thereafter (Fig. 2), although the relative proportion of TNFα mRNA in the cytokine milieu was still high up to day 5 post-treatment (Fig. 3). It has been shown that TNFα plays an integral role in the host defense against M. tb infection by activating macrophages and thus, contributing to induction and regulation of Th1-type cytokine and chemokine expression . By day 5 & 8 post-treatment, the antimicrobial response may have shifted to iNOS. The importance of cytokine-mediated up-regulation of macrophage expression of iNOS in the killing of M.tb has been clearly demonstrated in rodent models, but its contribution to the guinea pig model and human macrophages has been controversial [24, 25]. Prior to this study, our laboratory has only been able to detect iNOS mRNA transcripts under very limited circumstances. We detected iNOS mRNA transcripts in situ in pulmonary granulomas of non-vaccinated guinea pigs aerogenically challenged with virulent M. tb, and essentially none in BCG-vaccinated animals . Thus, there was an inverse relationship between iNOS expression and bacterial control, suggesting that it was the bacterial load in the tissue which drove iNOS mRNA expression. However, we demonstrate here that iNOS was detectable at very high levels in the guinea pig pleurisy model and its expression was associated with bacterial control (Fig. 1B & 2). Interestingly, the relative expression of iNOS transcripts was more prevalent in the pleural exudate than the surrounding tissue granulomas. This may be explained, in part, by the ability of cells and mediators to move more rapidly and freely in the pleural exudate compared to the granulomatous lesions. The contribution and importance of this cytokine to granuloma formation and bacillary control in guinea pigs remains speculative and must be examined more extensively. Studies using iNOS silencer genes in the guinea pig pleurisy model will help delineate the mechanisms by which iNOS contributes to bacterial control and granuloma maintenance.
In summary, neutralization of TNFα in our pleurisy model of TB hastened the progression of an anti-inflammatory response in the pleural exudates and associated granulomas. A concomitant decrease in bacillary loads demonstrated the minimal role for TNFα in bacterial killing in this model. This data are in contrast with previous findings by Chakravarty, et al whereby neutralization of TNFα in the mouse model resulted in enhanced expression of specific pro-inflammatory cytokines . The discrepancy between these findings can be explained, in part, by the route of infection and the animal model used. Our pleurisy model is induced in a previously vaccinated guinea pig. Therefore, our findings can be attributed, in part, to the effect of BCG vaccination as well as the neutralization of TNFα. While TNFα has been reported as being beneficial to the host and crucial for the control of M. tb infection, it is clear that it can also be detrimental when its expression is altered at certain phases of infection.
This work was supported in part, by the National Institutes of Health Grant RO1 AI-15495 to Dr. David N. McMurray and P30ES0910607 to the Center of Environmental and Rural Health at Texas A&M University.
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