Altered cellular infiltration and cytokine levels during early Mycobacterium tuberculosis sigC mutant infection are associated with late-stage disease attenuation and milder immunopathology in mice

Background Mouse virulence assessments of certain Mycobacterium tuberculosis mutants have revealed an immunopathology defect in which high tissue CFU counts are observed but the tissue pathology and lethality are reduced. M. tuberculosis mutants which grow and persist in the mouse lungs, but have attenuated disease progression, have the immunopathology (imp) phenotype. The antigenic properties of these strains may alter the progression of disease due to a reduction in host immune cell recruitment to the lungs resulting in disease attenuation and prolonged host survival. Results In this study we focused on the mouse immune response to one such mutant; the M. tuberculosis ΔsigC mutant. Aerosol infection of DBA/2 and SCID mice with the M. tuberculosis ΔsigC mutant, complemented mutant and wild type strain showed proliferation of mutant bacilli in mouse lungs, but with decreased inflammation and mortality in DBA/2 mice. SCID mice shared the same phenotype as the DBA/2 mice in response to the ΔsigC mutant, however, they succumbed to the infection faster. Bronchoalveolar lavage (BAL) fluid analysis revealed elevated numbers of infiltrating neutrophils in the lungs of mice infected with wild type and complemented ΔsigC mutant strains but not in mice infected with the ΔsigC mutant. In addition, DBA/2 mice infected with the ΔsigC mutant had reduced levels of TNF-α, IL-1β, IL-6 and IFN-γ in the lungs. Similarly, there was a reduction in proinflammatory cytokines in the lungs of SCID mice. In contrast to the mouse model, the ΔsigC mutant had reduced initial growth in guinea pig lungs. A possible mechanism of attenuation in the ΔsigC mutant may be a reduction in neutrophilic-influx in the alveolar spaces of the lungs, and decreased proinflammatory cytokine secretion. In contrast to mouse data, the M. tuberculosis ΔsigC mutant proliferates slowly in guinea pig lungs, a setting characterized by caseating necrosis. Conclusion Our observations suggest that the immunopathology phenotype is associated with the inability to trigger a strong early immune response, resulting in disease attenuation. While macrophages and T cells have been shown to be important in containing M. tuberculosis disease our study has shown that neutrophils may also play an important role in the containment of this organism.


Background
According to the latest WHO fact sheet, tuberculosis (TB) causes about 2 million deaths every year and 2 billion of the world's population is infected with Mycobacterium tuberculosis [1,2]. TB co-infection is also the number one killer of HIV patients which implicates the importance of a healthy immune system in controlling TB. M. tuberculosis is transmitted mainly by the respiratory route, and the primary site of infection is the lung. Following inhalation, these bacilli are phagocytosed by resident alveolar macrophages which recruit neutrophils, T cells and monocytes, and promote the local production of cytokines [3][4][5]. In vitro, M. tuberculosis triggers a Th1 type immune response which results in the release of TNF-α, IL-12 and IFN-γ [6,7]. These observations have been further substantiated in vivo using the mouse model [8][9][10][11][12]. While macrophages and T cells play key roles in the immune containment of M. tuberculosis, the role of other immune cells has received less attention. A recent study found that enhanced recruitment of neutrophils in lungs of some mouse strains is associated with increased susceptibility to M. tuberculosis infection [13], indicating that neutrophils may play a more important role during M. tuberculosis infection than previously thought.
Characterizing the exact roles for each of these components has been challenging and cytokine studies have produced conflicting results. IL-12 may be crucial in clearing M. tuberculosis infection in BALB/c mice but in a more resistant mouse strain, like C57BL/6, its effects are marginal [10]. IFN-γ is an important cytokine in controlling intracellular M. tuberculosis [8,11], but M. tuberculosisinfected-macrophages can secrete IL-6 which in turn prevents uninfected macrophages from responding to IFN-γ [14,15]. This negative feedback loop may enable the bacteria to persist in the host. Despite the inconsistencies, the host's immune system is a factor in determining the progression of the infection.
Antigenic properties of the different M. tuberculosis mutants also alter disease progression. Strains which can grow and persist in mouse lungs without eliciting severe damage have the immunopathology (imp) phenotype [16,17]. This phenotype results in reduced host immune cell recruitment to the lungs and prolonged host survival. Several imp mutants have been generated and tested in mice in order to characterize changes in the host tissues as well as in their immune responses. Some of these imp mutants include sigH, sigE, sigF, sigD, whiB3 and dnaE2 [16,[18][19][20][21][22][23]. In the case of the sigH mutant, four weeks after the mice were infected, there were fewer CD4 and CD8 T cells recruited to lung tissues in comparison to mice infected with the wild type strain [16]. The number of IFN-γ and TNF-α expressing CD4 T cells in these mice was also reduced. In order to determine if the imp phenotype can only be expressed in immunocompetent mice, SCID mice were infected with sigH, sigE, and sigD separately [16,20,21,23]. Results from these studies indicate that the imp phenotype appears to be dependent on functional cell-mediated immunity which the SCID mice lack.
To determine when these mutants begin expressing their phenotype in the host tissue, gene expression studies as well as RT-PCR were carried out. whiB3 [19,24] and sigH [16] for example were found to be maximally induced during the early phase of mouse lung infection while sigF [22] was highly expressed in stationary growth phase and was associated with late-stage disease in mice. Most of these studies focused on bacterial survival in the host rather than the host's immune response. However, these mutants could reveal how the host immune system contains M. tuberculosis infection.
The RNA polymerase sigma factors of M. tuberculosis, such as SigC, confer DNA binding at specific promoter sites and play a role in transcription initiation. In a previous study we found that the ΔsigC mutant had an imp phenotype where the mutant persists in mouse lungs but fails to cause mortality as rapidly as the wild-type. SigC was found to be essential for lethality of M. tuberculosis in mice [20]. To address the question of host factors, we infected mice with the ΔsigC mutant and assessed cell infiltration patterns and cytokine levels during different time points after aerosol infection. We found that mice infected with the wild type M. tuberculosis strain had higher levels of inflammatory cytokines and infiltrating neutrophils when compared to the ΔsigC mutant infected mice. The same observation was also noted when SCID mice were exposed to the ΔsigC mutant strain. We also characterized the pattern of M. tuberculosis ΔsigC mutant infection in guinea pigs and found dramatically reduced bacterial proliferation in contrast to the mouse pattern. Our observations suggest that the imp phenotype is associated with the inability to trigger a strong early immune response, which may result in disease attenuation.

M. tuberculosis ΔsigC mutant infection in immunocompetent mice
We infected three groups of mice separately with the ΔsigC mutant, the ΔsigC complemented mutant, and the wild type parental strain, CDC1551. Bacterial growth in the lungs of DBA/2 mice was observed to increase one week post infection and to plateau at week 4 for both the CDC1551 and    figure 1C; d, e and 1f). These observations indicated that milder lung pathology, secondary to reduced bacterial growth, can contribute to host survival.

Differential cellular infiltration in alveolar spaces seen with the ΔsigC mutant
The role of resident alveolar macrophages in eliminating invading bacteria in the lung has been widely accepted [25,26]. However, less is known regarding the role of polymorphonuclear cells in pulmonary tuberculosis. Thus, we collected BAL fluid and employed cytology to classify which cell types were recruited into the alveolar spaces during disease progression in wild type versus ΔsigC mutant infection. The total number of recovered cells from BAL varied between less than 10 5 cells in noninfected mice to 1 million cells at days 14 and 70 (figure 2). Throughout this study, the number of BAL cells from mice infected with the ΔsigC mutant remained on average less than 5 × 10 5 cells per mouse in contrast to mice infected with the CDC1551 and the complemented ΔsigC mutant (figure 2) whose counts were ~1 × 10 6 . The lymphocyte population, which consisted of B, T and NK cells, remained at a relatively constant level throughout the study. Three cell types were detected via the cytospin assay: lymphocytes, monocytes/macrophages and neutrophils. The majority of BAL cells were alveolar macrophages at about 90%, while lymphocytes and neutrophils made up the remaining cell population. As time pro-gressed, more neutrophils were found in the BAL fluid of both the complemented ΔsigC mutant and CDC1551 infected groups (figure 2). At days 14 and 70, neutrophils became the main cells in the lungs of these two groups, but this was not observed in the lungs of ΔsigC infected mice. Instead, decreased numbers of infiltrating neutrophils were observed at day 70 and day 120. . TGF-1β, IL-4 and IL-10 (the latter two not shown) also exhibited the same profile indicating that these cytokines may be undetectable or inactive in the lungs during this study period.

M. tuberculosis ΔsigC mutant infection in SCID mice
In DBA/2 mice infected with the ΔsigC mutant, lower numbers of infiltrating neutrophils coincided with DBA/2 mouse survival score and CFU counts

Accelerated bacterial growth coincides with increased tissue damage in SCID mice
Lungs from the CDC1551 and the complemented mutant groups developed severe tissue damage but the damage was much milder for the ΔsigC mutant group. Lung sections analyzed from ΔsigC mutant infected mice had fewer lesions 28 days (figure 6) post infection in contrast to both the complemented mutant and CDC1551 infected groups. Ziehl-Neelsen stainings confirmed the different bacillary burden in the lungs of the three groups ( figure  6). However at time of death (75-90 days post infection) the lungs of the ΔsigC mutant group were as severe as both the CDC1551 and complement control groups (data not shown).

Discussion
M. tuberculosis mutant strains which exhibit the imp phenotype and show milder lung pathology as well as prolonged host survival in comparison to the wild type strain have been described [16,18-23]. In our current survival study, gross pathology and histopathology revealed the mild pathology with the ΔsigC mutant consistent with this reduced pathology pattern. On the other hand, the lung CFU counts of mice infected with the ΔsigC mutant strain, while relatively high, began to lag three weeks post infection and remained between 1 to 1.5 log 10 units lower than the CDC1551 and ΔsigC complemented mutant strains.
In contrast to earlier studies of the ΔsigC mutant [20], the lung CFU count showed relative containment compared with the wild type; this occurrence could play a role in the relative reductions in some of the inflammatory parameters observed with the mutant. The major cell types infiltrating the alveolar spaces of the CDC1551 and ΔsigC complemented mutant strain infected mice were neutrophils, monocytes and lymphocytes. Interestingly, the numbers of infiltrating neutrophils in the BAL of ΔsigC mutant infected mice were at least 10-fold less than the other two groups. Reduced levels of TNF-α, IL-1β, IL-6, IL-12, IFN-γ, CCL-2 and CCL-5 were also detected in the lung homogenates of ΔsigC mutant infected mice. However, the lung is a separate compartment from the BAL and the levels in one may not necessarily relate to the other. A.

SCID mouse survival score and lung CFU counts
These cytokines are commonly associated with the inflammatory environment of M. tuberculosis infection [3,4,7,[9][10][11][12][33][34][35][36][37]. We did not examine the role of the adaptive immune system in clearing M. tuberculosis infection. However, previous studies have placed a great emphasis on the role of a T helper 1 (Th1) type response, involving the engagement of CD4 + and CD8 + effector T cells and the production of macrophage-activating cytokines like IFN-γ, TNF-α and IL-12. Together with T cells and NK cells, macrophages have been shown to secrete TNF-α, IL-12 and IFN-γ during intracellular infections [38]. IFN-γ is an important mediator of macrophage activation and regulation of this cytokine has been considered crucial in combating infection [8,11]. IL-12, which induced IFN-γ production by lymphoid cells [39,40], did not change significantly in this study. This may be because we focused on the primary site of infection, i.e., the lung tissue homogenates, and not on lymphoid tissues. The lack of neutrophil infiltration and reduced levels of pro-inflammatory cytokines like TNF-α, IL-1β, IL-6 and IFN-γ in the lungs of ΔsigC mutant infected mice may explain the milder disease in the DBA/2 and SCID mice. However, in SCID mice a significant difference in cellular infiltration in the BAL was not observed. Since neutrophils have been identified in tuberculous lung granulomas in humans, guinea pigs and mice [41], their role in granuloma formation has been suggested [30, 42,43]. Neutrophils have been shown to produce different chemokines which are crucial for bacterial clearance [44,45]. Yet the levels of both CCL-2 and CCL-5 in the lungs of ΔsigC mutant infected mice were marginal and might not be involved in the recruitment of neutrophils in this study.
Both IL-1β and IL-6 are involved in the host immune response to M. tuberculosis infection and are produced by monocytes, macrophages, dendritic cells and neutrophils [46]. IL-1β was detected in lung sections of tuberculosis patients [47,48] and in the animal models [49,50]. This cytokine has been postulated to play a role in restricting the spread of the bacillary growth after M. tuberculosis infection by forming granulomas [51,52]. Our data would support this hypothesis because we saw severe granuloma-like lesions in the lungs of the CDC1551 and ΔsigC complemented mutant groups but not among the ΔsigC mutant infected mice where a lower concentration of IL-1β was detected.
Guinea pigs form caseating granulomas in their lungs in response to M. tuberculosis infection while infected mice do not. In this study, ΔsigC mutant infected guinea pigs had undetectable CFU counts 4 weeks after infection in contrast to both the CDC1551 and the ΔsigC mutant complemented infected groups. A recent study compared the survival of identical pools of M. tuberculosis mutants in both animal species after aerosol infection and found that M. tuberculosis genes required for survival were cleared earlier in guinea pigs than in mice [53]. Differences in host response to the invading bacilli may be species specific and the ΔsigC mutant strain can express a different pheno-Lung sections from SCID mice 28 days post infection    B.
type in guinea pigs than in mice. Here we showed that the ΔsigC mutant strain proliferated poorly with a growth in vivo (giv) defect in guinea pigs. A similar finding was also observed when guinea pigs and mice were infected with the M. tuberculosis ΔdosR mutant strain [54]. Similar findings have been recently reported in a study of several sigma factor mutants evaluated in the guinea pig model [55].
The major findings in this work are the low influx of neutrophils and reduced levels of known pro-inflammatory cytokines which are associated with M. tuberculosis infection in the BAL of mice exposed to the ΔsigC mutant strain. We selected the DBA/2 mouse strain for our study because of it susceptibility to M. tuberculosis infection hence fewer bacilli were required to induce the disease [56]. These mice survived for more than one year after aerosol infection with the ΔsigC mutant strain and immunological analysis revealed that the attenuated mutant strain was not as immunogenic as both the wild type and complemented strain. Our data are similar to those in a recent study looking at the immunological changes of susceptible mouse strains to M. tuberculosis (H37Rv) infection [13]. In that study, elevated numbers of neutrophils in the BAL correlated with higher susceptibility to succumbing to M. tuberculosis infection. However, our study is the first to show that neutrophil infiltration can be affected by an attenuated M. tuberculosis mutant strain. Indeed, lipid heterogeneity, rather than genetic variations, is believed to be the major cause of differences in pathogenicity between strains [57,58]. Transcriptional profiling of the ΔsigC mutant versus the wild type strain [20] revealed that genes involved in biosynthesis of cofactors, cell wall components, fatty lipids, energy metabolism, protein folding and stabilization, and protein synthesis were downregulated. Hence it will be of interest to investigate whether such changes in the cell envelope may contribute to the expression of the imp phenotype of the ΔsigC mutant strain in mice.

Conclusion
We have found that the ΔsigC mutant with the imp phenotype is unable to trigger a strong early immune response. This may occur due to a low influx of neutrophils and reduced levels of pro-inflammatory cytokines. Our study is the first to show that neutrophil infiltration can be affected by an attenuated M. tuberculosis mutant strain.

Bacterial strains and culture conditions
The sigC mutant was constructed as described [20]. M. tuberculosis CDC1551 wild type, ΔsigC deleted mutant and the ΔsigC deleted complement mutant were cultured in Middlebrook 7H9 (liquid medium containing 0.05% TWEEN 80 and supplemented with OADC and 0.2% glyc-erol) at 37°C and 0.5% CO 2 . At the appropriate times OD readings at 600 nm were taken to determine bacterial density.

Mouse virulence assays
Female DBA/2 and CB17-SCID mice were infected by the aerosol route by placing liquid inoculum into the nebulizer of a Glass-Col aerosol machine and running the standard program. Their lungs were implanted with ~100 bacteria per lung at day 0 of the experiment. At day 1, lungs were collected separately and homogenized and dilutions plated onto 7H10 agar plates to confirm that comparable numbers of bacteria were implanted per mouse lung. The bacteria were incubated at 37°C and the colony forming units (CFU) determined.

Guinea pig infection
Female outbred Hartley guinea pigs (~500 g) were infected using a Madison aerosol generation chamber calibrated to deliver approximately 50 to 100 bacteria per lung at day 0. Three to five animals were sacrificed 4 and 8 weeks post infection and their entire lungs homogenized for CFU counts.

Cytokine Assay
Supernatants from homogenized lungs were collected and passed through a 0.22 μM Millipore filter. Using the Duo-Set ELISA kits from R&D Systems, (Minneapolis, MN) supernatants were tested for the presence of CCL-2, CCL-5, TNF-α, IL-6, IL-1β, IL-4, IL-12p70 and TGF-β1. IL-10 and IFN-γ sandwich ELISAs were performed using reagents from MabTech (Cincinatti, OH). Cytokine assays were conducted according to the instructions provided by the manufacturers.

BAL and Cytosmear
Bronchoalveolar lavage cells (BAL) were harvested by exposing the trachea and excising a small incision using a scissor. A blunt needle connected to a 1 ml syringe is inserted into the cut and ice cold PBS is gently flushed into the trachea and lungs. BAL cells were washed twice at 1200 RPM for 7 min at 4°C and resuspended in PBS with 1% FCS. 100 μl of BAL cells was mounted on a glass slide with cytospin centrifugation (StatSpin Cytofuge 2) and sealed. Slides were air dried overnight and stained with Protocol HEMA 3 stain set and differential cell counts performed.

Histopathology
Lungs were excised from all animals and stored in 10% formalin, embedded and stained with haematoxylin and eosin (H&E) for pathological analysis or with Ziehl-Nielsen stain to evaluate the presence of acid fast bacilli in these organs.

Statistical analysis
The GraphPad Prism 4 software program was used to perform all statistical analysis (GraphPad Software Inc., San Diego, CA). Kaplan-Meier analysis was used to determine statistical significance of the differences in survival of mice. The statistical significance of three-way comparisons was tested by two-way analysis of variance (ANOVA) with Bonferroni post-tests.