Small Animal Model of Post-chemotherapy Tuberculosis Relapse in the Setting of HIV Co-infection

Abstract

Tuberculosis relapse following drug treatment of active disease is an important global public health problem due to the poorer clinical outcomes and increased risk of drug resistance development. Concurrent infection with HIV, including in those receiving anti-retroviral therapy (ART), is an important risk factor for relapse and expansion of drug resistant Mycobacterium tuberculosis (Mtb) isolates. A greater understanding of the HIV-associated factors driving TB relapse is important for development of interventions that support immune containment and complement drug therapy. We employed the humanized mouse to develop a new model of post-chemotherapy TB relapse in the setting of HIV infection. Paucibacillary TB infection was observed following treatment with Rifampin and Isoniazid and subsequent infection with HIV-1 was associated with increased Mtb burden in the post-drug phase. Organized granulomas were observed during development of acute TB and appeared to resolve following TB drug therapy. At relapse, granulomatous pathology in the lung was infrequent and mycobacteria were most often observed in the interstitium and at sites of diffuse inflammation. Compared to animals with HIV mono-infection, higher viral replication was observed in the lung and liver, but not in the periphery, of animals with post-drug TB relapse. The results demonstrate a potential role for the humanized mouse as an experimental model of TB relapse in the setting of HIV. Long term, the model could facilitate discovery of disease mechanisms and development of clinical interventions.

Keywords: HIV; TB; TB and HIV co-infection; TB chemotherapy; TB relapse; immune response; pathology; tuberculosis.

Figure 1

Paucibacillary TB and granuloma resolution following TB chemotherapy. (A) Experimental design for study 1. HIS BLT mice were generated and infected i.n. with 10² CFU of Mtb H37Rv for 8 weeks followed by treatment with RIF and INH by oral gavage (750 μg/day) for 8 weeks. Drug treatment was terminated and HIS mice were assigned to i.v. infection with 2,500 TCID₅₀ of HIV-1 (JR-CSF) or mock (PBS) using 3 mice per group. Relapse of TB was assessed at 8 weeks post-HIV infection. Infection with Mtb and drug efficacy was confirmed by CFU enumeration in tissues of 2 animals per designated (*) time point with a limit of detection of 30 organisms. Representative images from histological findings as visualized with H&E and Ziehl-Neelson staining and brightfield microscopy. (B) Granulomatous pathology characteristic of active TB in HIS mouse lung and (C) corresponding AFB present in the inflamed tissue. (D) Healthy lung from a non-infected mouse. (E) Mycobacterial burden in the lung, as determined by CFU enumeration following 8 weeks of infection and 8 weeks of drug treatment with RIF and INH. (F) Residual interstitial inflammation observed in lung following drug treatment, including (G) AFB negative inflammatory cells frequently observed in interstitium, and (H) Rare AFB observed in alveolar macrophage.

Figure 2

Pulmonary TB relapse in HIS mice co-infected with HIV-1. Pulmonary TB relapse outcomes in the setting of HIV infection in study 1. Following establishment of paucibacillary Mtb infection with chemotherapy, HIS mice were co-infected i.v. with mock (PBS) or HIV-1 (2,500 TCID₅₀ JR-CSF). Experiments were terminated at 8 weeks post HIV or mock infection to assess TB relapse. (A) Lung bacterial burden during the relapse phase in TB and TB/HIV infection groups. Histological appearance of lung as shown with representative images from animals in both TB and TB/HIV groups. (B) Non-remarkable lung with occasional pockets of inflammation and frequent areas of interstitial inflammation that include features illustrated in C-H. (C) Foamy alveolar macrophages that are (D) lacking AFB. (E) Paraseptal and interstitial inflammation with (F) occasional AFB positive (arrowhead) inflammatory cells in interstitium. (G) Larger areas of inflammation containing (H) pockets of AFB (arrowhead). Data shown in (A) is the mean ± SEM and statistically significant differences between treatment groups are shown as *p < 0.05.

Figure 3

Pulmonary TB relapse following high dose Mtb exposure and HIV infection (A) Experimental overview for study 2. HIS BLT mice were infected i.n. with an estimated 10³ CFU of Mtb H37Rv for 4 weeks and subsequently treated with RIF and INH by oral gavage (750 μg/day) for 8 weeks. Drug treatment was terminated and HIS mice were assigned to i.v. infection with 2,500 TCID₅₀ of HIV-1 (JR-CSF) or mock (PBS) using 4 HIS mice per group. Relapse of TB was assessed at 5 weeks post-HIV infection due to observation of signs of disease (e.g., weight loss). *Denotes confirmation of infection and drug activity in selected animals. (B) Confirmation of infection and drug efficacy prior to, during, and at the completion of TB chemotherapy (n = 4, with 1 animal per time point). (C) Bacterial burden 5 weeks post-HIV or mock infection in the TB and TB/HIV groups. Histological appearance of tissue from HIS mice following Mtb infection, drug treatment with RIF/INH, and at TB relapse is shown in (D–K). (D) Granulomatous inflammation at 4 weeks post-infection with Mtb and (E) abundant AFB positive areas within the granuloma. (F) Residual interstitial inflammation and (G) rare AFB (arrowhead) in inflammatory cells in the interstitium, after 8 weeks of RIF and INH. Similar histological appearance of lung following relapse in TB and TB/HIV experimental groups is shown with representative images in (H–K). (H) Frequently observed areas that include non-remarkable lung, interstitial inflammation, and perivascular inflammation and (I) AFB in small pockets of inflammation. (J) Infrequent granulomatous lesion and (K) AFB in a granuloma center.

Figure 4

Hepatic TB relapse in the setting of HIV co-infection. Infection, drug treatment and TB relapse outcomes in liver of HIS mice given high dose (10³ CFU) Mtb infection in study 2. (A) CFU in the liver 4 weeks after i.n. infection with Mtb and after 2, 4, and 8 weeks of TB chemotherapy with RIF/INH. (B) Inflammatory foci containing (C) clusters of AFB (arrowheads) in the liver of mice following infection with Mtb. (D) Lesions with frequent multi-nucleated giant cells (arrowheads) observed following 8 weeks of TB chemotherapy which (E) lack detectable AFB. (F) Hepatic mycobacterial burden (CFU) in the TB relapse phase in HIV mono-infected, and Mtb and drug treated mice with mock (TB) or HIV (TB/HIV) co-infection. (G) Inflammatory foci similarly evident in liver from both TB and TB/HIV relapse in which (H) rare AFB are observed in the TB relapse phase. Data in B are means ± SEM with statistically significant differences between treatments shown as *p < 0.05.

Figure 5

HIV replication in lung and liver during TB relapse. Viral load and distribution of HIV+ cells in the periphery and tissues in the TB relapse phase in study 2. (A) Viral load in the plasma, lung, and liver as measured by ELISA (Zeptometrix) specific to HIV p24 capsid protein. (B) Detection of a cellular syncytia (arrowhead) characteristic of HIV infection as observed in bright field microscopy following visualization with H&E. (C–G) HIV infected cells were detected with RNA-Scope specific to HIV gag. (C,D) HIV+ cells in the lung interstitium and near portal tracts in the liver of a HIS mouse with HIV mono-infection. (E–G) HIV+ cells were observed in inflamed interstitium (E) and in the periphery of TB lesions (F), as well as near blood vessels in the liver (G), of TB/HIV co-infected mice at TB relapse. Data shown in (A) are means ± SEM with statistically significant differences between indicated treatment groups shown as ***p < 0.001.

Figure 6

Pulmonary immune microenvironment of TB and TB/HIV relapse. Lung supernatants were harvested from disrupted lung tissue to assess differences in the immune microenvironment at TB relapse, in the presence or absence of HIV. Analysis of lung cytokines and chemokines was performed with a human multiplex ELISA (Bio-rad Bio-plex Pro™ human cytokine 27-plex kit. Shown are results from selected cytokines or chemokines at the TB relapse endpoint from study 2. Data are the means ± SEM with statistically significant differences compared to non-infected controls designated as follows: *p < 0.05. Significant differences between the indicated infection groups are shown as #p < 0.05; ##p < 0.01.