*3.4. rBCG-N-hRSV Immunization Promotes a Higher Induction of HO-1 on Epithelial Cells than in DCs at Late Infection Times*

BCG is commonly used as a vaccine to prevent infections by *M. tuberculosis* and is often used as a vector for generating new recombinant vaccines [14]. Therefore, we decided to evaluate if the immunization with an rBCG-N-hRSV can modulate the expression of HO-1 in hRSV-infected mice. After 7 and 14-dpi with hRSV, mice were euthanized, and both epithelial and DCs from the lungs were stained to evaluate the presence of HO-1 (Figure 5). The HO-1 expression observed on day 7 post-infection in DCs showed a non-significant decrease in the *rBCG-N-hRSV* immunized mice compared to mock-treated mice. A similar result was observed in the rBCG-N-hRSV group in *BCG-WT* and hRSVinfected mice (Figure 5B). In contrast, when HO-1 was evaluated in epithelial cells, the mock-treated group showed the lowest non-significant levels of expression as compared to the rest of the infected groups (*mock-treated* compared to *hRSV-infected* (*p* = 0.9680), *BCG-WT* (*p* = 0.2232), *rBCG-N-hRSV* (*p* = 0.733)) (Figure 5C). When the HO-1 expression was evaluated on day 14 post-infection in DCs, similar results were obtained compared to day 7 post-infection. Interestingly, although the behavior of the groups was similar to that previously reported, the magnitude order was 4 to 5-fold less (Figure 5D). On the other hand, epithelial cells showed a different effect in the HO-1 expression on day 14 postinfection, demonstrating a different behavior distinct from the HO-1 expression in epithelial

cells on day 7 post-infection. In this line, the HO-1 expression was higher in both *mock-treated,* and *rBCG-N-hRSV* immunized mice than in hRSV-infected or *BCG-WT* groups (Figure 5E), suggesting that the immunization with rBCG-N-hRSV promotes a lasting induction of HO-1 through time.

**Figure 5.** Detection of Heme Oxygenase (HO)-1 in cell populations during an immunization scheme with recombinant BCG expressing the nucleoprotein of human respiratory syncytial virus (rBCG-N-hRSV). Immunization scheme of BALB/c mice (**A**). Analysis by flow cytometry of dendritic cells (DC) (**B**) and epithelial cells (**C**) at 7 days post-challenge, and DCs (**D**) and epithelial cells (**E**) at 14 days post-challenge. Data are shown as means ± SEM of one independent experiment with three animals per group. One-way ANOVA was performed with a post-hoc Tukey test.

#### **4. Discussion**

The infection with hRSV is the principal cause of ALRTI, and it has been demonstrated that it can predispose to secondary lung infections [1]. The effects of primary viral infections on the outcome of concomitant secondary acute bacterial infections have only been studied in mice [13], such as *S. pneumoniae,* which induces a more severe pulmonary pathology [11,44]. Some studies have demonstrated that the immunopathology generated during coinfections of viruses, with either other viruses or bacteria, depends on the primary viral infection stage [14,42]. Therefore, it is important to establish the stage of the viral infection that will be evaluated. It has been reported that the administration of *S. pneumoniae* following the peak of hRSV replication in the lungs increases bacterial

burden (analyzed as CFU and infiltration of inflammatory cells [13]. However, the link between hyperresponsiveness of the airways and long-term pulmonary sequels is not clear, although two possible explanations have been suggested. First, it was proposed that hRSV produces long-term damage in the airway epithelium, which may favor the infection by other pathogens [13,45]. In this line, studies with other respiratory viruses, such as influenza A virus, have shown a detrimental effect of the viral infection on the survival rate and clearance of *M. tuberculosis* in mice after viral clearance was demonstrated in the lungs [46]. As a second hypothesis, it was proposed that hRSV-infection exerts an immunomodulatory effect on the lungs, which predisposes to allergy and asthma [47]. To the best of our knowledge, no studies address whether hRSV infection exerts longlasting effects on pulmonary immune responses, especially after the resolution of the initial viral insult.

As mentioned previously, the infection with BCG is a valuable model for studying antimycobacterial immunity, including forming granulomatous lung lesions and acquiring a paucibacillary state [15,16]. The mouse model of intranasal BCG infection is suitable for addressing whether hRSV blunts the desired type 1 T helper (Th1)-driven anti-mycobacterial immune responses, which correlate with protection to other mycobacterial species [48,49]. Furthermore, it has been described that hRSV-effects can last up to 42 dpi in the BALB/c mouse model [50] and up to 20 dpi in C57BL/6 mice [51].

This work aimed to unravel whether a previous infection with hRSV can elicit an immune response that facilitates the mycobacterial infection in a murine model. Therefore, we used an intranasal administration of attenuated *M. bovis* strain, BCG, to establish our subsequent infection model. Since BCG has been used as an infection model of TB [49,52,53], here we used an intranasal administration of BCG after the clearance of the infection with hRSV in mice to elucidate if this viral infection promotes an immune response that favors a secondary BCG infection. One of the significant discoveries in this work was the detection of brown structures in the lung epithelium in the *hRSV-BCG* group, where it was possible to identify the presence of mycobacteria in the acid-fast staining (Figure 3A). One hypothesis is that these brown spots, present only in the *hRSV-BCG* group, are lipid droplets that lose the acid-fast staining, as the color is brown and even considered orange or red [42] (Figure 3A and Supplementary Figure S3A). These drops of lipids can result from a mechanism of *Mycobacterium* when it remains dormant, considering that the *Mycobacterium* is surrounded by a monolayer of phospholipids and uses these structures as a primary source of carbon [42]. This stage occurs when the bacteria are in a context that promotes hypoxia, such as when the pulmonary tissue is not yet fully recovered during respiratory infections, such as hRSV [54,55]. The absence of complete recovery in the pulmonary tissue can be suggested due to the significant increase of CD200 (Figure 1F) and HO-1 relative expression (Figure 1E), which promote an anti-inflammatory environment [55]. Additionally, we can suggest that both CD200 and HO-1 might play a role in restructuring the lung's epithelial environment after 10 dpi with hRSV [55]. After inoculating the mice with BCG, further damage was observed, and the cell infiltration remained increased, as shown in Figure 2. Similar results are observed in Supplementary Figure S2. The mycobacteria acquire a dormant state that increases the expression of HO-1 to produce carbon monoxide and promote an anti-inflammatory state in the host cells [23,56]. Accordingly, the increased detection of HO-1 and the possibility of macrophages containing bacilli in a dormant state (Figure 3) can be related to the presence of mycobacteria in the epithelium [57,58]. In this context, tissue oxygenation is impeded, favoring the conditions in which the *Mycobacterium* is put into a dormant state through triglyceride metabolism [59–61]. Unlike hRSV, which produces ALRTI, BCG infection produces a less diffuse pulmonary pathology and, therefore, a less pronounced body weight loss (Figure 2B,E).

One of the limitations of this study was that the observation of mycobacterial infection was performed over an unprolonged period. With an even longer-term scheme, it would be possible to observe how the mycobacteria behave at cellular levels and if the cellular populations change on this day. This is because the airway epithelium has a cell replacement every twenty days but not in the alveolar macrophages, which can stay in the lung for years [62–64]. However, it must be considered that the alveolar macrophages will remain in the lungs for long periods if they are not destroyed by pathogenic infections [65]. Even more, some respiratory viruses, such as influenza virus, can promote changes in the alveolar macrophages to induce an extended antibacterial response [66]. In addition, more days allow for identifying the mycobacteria by acid-fast staining, which is correlated with the CFUs counts (Figures 2F and 3A). This study also used BCG mycobacteria as a model, making it possible to use it in a murine model and address scientific questions with an attenuated pathogen. However, this work proves that it serves as a model for research on secondary *Mycobacterium* infections despite not having the same effect and clinical symptoms as an infection with *M. tuberculosis*.

In addition, relative cytokine expression was evaluated for *ifn-γ*, *ifn-β*, and *il-6* (Figure 4B–D), which participate in the control of mycobacterial infections and other bacterial infections [67,68]. Thus, *ifn-γ, ifn-β,* and *il-6* are related to mycobacteria colonization in these schemes. In the case of *ifn-γ* in the short scheme of infection, the relative expression was more elevated in the *hRSV-BCG* group than in *Mock-BCG* and the other groups (Figure 4B). Interestingly, in the short infection scheme, the hRSV-BCG group had a lower relative expression of *ifn-β* than the *hRSV-Vehicle* (Figure 4C). Still, in the long infection scheme, only the *hRSV-BCG* group had a higher relative expression of this gene than the *hRSV-Vehicle* (Supplementary Figure S4C). *ifn-β* is a molecule that helps delay the beginning of mycobacterial infection [67], and as such, the data observed on the long scheme might be explained due to viral clearance. However, in the long scheme, we can see a similar phenomenon to *ifn-γ*, where the group with the highest expression was in the groups with mycobacteria inoculation (Supplementary Figure S4B), which could be attributed to the presence of mycobacteria. This increase in the expression of *ifn-γ* could be associated with the presence of alveolar macrophages, which are sentinels in the pulmonary epithelium that promote a favorable environment [69,70]. Both the short (Figure 4D) and long (Supplementary Figure S4D) schemes presented similar relative expressions of *ifn-γ*. Lastly, the increased relative expression of *il-6* does not inhibit the growth of mycobacteria, even though it has been reported that an increased secretion helps to protect the host against mycobacteria [68]. These data would imply that the presence of mycobacteria activates the expression of these cytokines.

We have previously shown the anti-inflammatory effect of HO-1 induction in vivo and in vitro [20]. Here we extend these observations by showing that at 7 dpi with hRSV, mice pre-vaccinated with BCG showed an elevated expression of HO-1 in DCs, but the group treated with mock had the highest HO-1 within hRSV and BCGs-groups (Figure 5B). A similar effect was found at day 14 dpi, but the activation of HO-1 was four to five-fold less than previously reported (Figure 5D), and this HO-1 in DCs elevated just in the mock group with respect to hRSV-infection was reported [20]. Interestingly, the effect of HO-1 in epithelial cells differed from that observed in DCs. Here, at 7 dpi, all hRSV-infected groups had a similar HO-1 response to the mock-treated group (Figure 5C). However, this effect was different at 14 dpi, where only rBCG-N-hRSV immunized mice had high HO-I levels, similar to the mock-treated group (Figure 5E). This last statement might suggest that hRSV-N-BCG promotes a prolonged state of HO-I activation in epithelial cells that helps eliminate the virus and recover damaged tissue [71]. No changes in the MFI of HO-I were found between the groups at different times (data not shown). Interestingly, it has been reported that the infection with hRSV can modify the methylation profile of immune cells and epithelial cells, promoting the secretion of Th2 cytokines and viral replication [72]. Since the activation of Nrf2 can be regulated epigenetically, it would be interesting to evaluate whether hRSV infection can modulate the activation of Nrf2 in an epigeneticallymanner [73]. In this sense, a limitation of our article is that we did not explore whether a primary infection with hRSV can modulate epigenetic changes that may participate in the resolution of the disease against subsequent administration with *M. bovis*.

#### **5. Conclusions**

Pulmonary infections modulated by hRSV negatively modulate the respiratory and immune response, which promotes a sub-sequential *M. bovis* exposure, leading to inefficient mycobacterial clearance and increased host inflammatory response. The clearance is impeded by a possible dormancy state established by mycobacteria. Additionally, the mycobacteria promote HO-1 expression activating the dormancy state. In other words, hRSV promotes the development of pulmonary-tuberculosis-like in mice by increasing lung inflammation and the survival of infecting bacilli.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/antiox11081453/s1, Figure S1: Gating strategy for identifying pulmonary cells found either in bronchoalveolar lavage (BAL) or in lung homogenates; Figure S2: Evaluation of infection parameters from primary infection with human respiratory syncytial virus (hRSV) and 21 days post-infection (dpi) with Bacillus Calmette-Guerin (BCG). (A) Scheme of infection with a prime infection of hRSV A2 (1 <sup>×</sup> <sup>10</sup><sup>7</sup> plaque formation units (PFU) and then a subsequent instillation of BCG at 21 dpi. (B) Percent body weight in C57BL/6 mice background after 21 days of subsequent BCG challenge, the mice have a prime infection with hRSV. (C) Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) for determination of viral load. Determination of infiltration in the lungs of mice infected with BCG on day 21 post-infection with hRSV. (D) Lung tissue sections were stained with Hematoxylin and Eosin (10X magnification). (E) Histopathological score. (F) Bacterial load in the BCG-groups. (G-J) Flow cytometry analyses of BAL from mice infected with *Mycobacterium bovis (M. bovis).* The figure shows the absolute cell numbers for neutrophils (G), monocytes (H), eosinophils (I), and alveolar macrophages (J) in BAL of BCG-infected mice. Data are shown as means ± SEM of three independent experiments with 3–4 animals per group. One-way ANOVA was performed with a post hoc Tukey test. (\*\* *p* < 0.01; \*\*\*\* *p* ≤ 0.0001). (F) t-student test was using parametric distribution was used; Figure S3: The long scheme of hRSV-infection favors colonization infection with mycobacteria in the lungs. (A) Acid-fast staining of attenuated *M. bovis*-infected mouse lungs that were collected on day 21 post-infection with hRSV (10X and 100X magnification). Quantification by RT-qPCR of Heme Oxygenase *(ho)-1* (B) and the nuclear factor erythroid 2-related (*nrf2*) (C). Data are shown as means ± SEM of three independent experiments with 3–4 animals per group. One-way ANOVA was performed with a posterior Tukey test; Figure S4: Determination of the relative expression of immunomodulatory molecules and cytokines in infection with BCG at 21 days post-hRSV-infection. Quantification by RT-qPCR of the OX-2 glycoprotein membrane (*cd200*) at day 10 post-infection (A), interferon gamma (*ifn-γ*) (B), interferon beta (*ifn-β*) (C), and interleukin (*il)-6* (D). Data are shown as means ± SEM of three independent experiments with 3–4 animals per group. One-way ANOVA was performed with a posterior Tukey test.

**Author Contributions:** G.C.-M. performed experiments, designed the study, analyzed data, and wrote and revised the manuscript. J.A.S. performed experiments and manuscript revision. C.A.A. participated in manuscript revision and editing. S.M.B. supported in manuscript revision. A.M.K. is the leading investigator and supported in the organization, experimental design, and full manuscript revision. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by FONDECYT 1070352; 1190830, FONDEF D061008, FONDEF D11I1080, PAI ANID No. SA77210076, PAI ANID No. SA77210051, ANID/CONICYT scholarship 21210662, and the Millennium Institute on Immunology and Immunotherapy CN09\_016/ICN 2021\_045; former P09/016-F.

**Institutional Review Board Statement:** All experimental protocols followed guidelines from the Sanitary Code of Terrestrial Animals of the World Organization for Animal Health (OIE, 24. Edition, 2015) and were reviewed and approved by the Scientific Ethical Committee for Animal and Environment Care of the Pontificia Universidad Católica de Chile (Protocol number 160915010 and 160405005). All mouse experiments were conducted in agreement with international ethical standards and according to the local animal protection law number 20,800.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data generated during this study are readily available in the manuscript.

**Acknowledgments:** We thank our veterinarian María José Altamirano for her support in handling and caring for the experimental animals and evaluating the histopathological score. We also thank Francisco Salazar for technical assistance. We also thank Pablo Cespedes (CONICYT/FONDECYT POSTDOCTORADO No. 3140455) for technical assistance and experimental design. Finally, we thank Biorender for partial material used for the design of some figures.

**Conflicts of Interest:** The authors declare no conflict of interest.
