Abstract
Infection with Mycobacterium tuberculosis (Mtb) results in the primary formation of a densely packed inflammatory foci that limits entry of therapeutic agents into pulmonary sites where organisms reside. No current therapeutic regimens exist that modulate host immune responses to permit increased drug penetration to regions of pathological damage during tuberculosis disease. Lactoferrin is a natural iron-binding protein previously demonstrated to modulate inflammation and granuloma cohesiveness, while maintaining control of pathogenic burden. Studies were designed to examine recombinant human lactoferrin (rHLF) to modulate histological progression of Mtb-induced pathology in a non-necrotic model using C57Bl/6 mice. The rHLF was oral administered at times corresponding to initiation of primary granulomatous response, or during granuloma maintenance. Treatment with rHLF demonstrated significant reduction in size of primary inflammatory foci following Mtb challenge, and permitted penetration of ofloxacin fluoroquinolone therapeutic to sites of pathological disruption where activated (foamy) macrophages reside. Increased drug penetration was accompanied by retention of endothelial cell integrity. Immunohistochemistry revealed altered patterns of M1-like and M2-like phenotypic cell localization post infectious challenge, with increased presence of M2-like markers found evenly distributed throughout regions of pulmonary inflammatory foci in rHLF-treated mice.
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Acknowledgements
This work was given in part at the 14th International Conference on Lactoferrin Structure, Function and Applications, held in Lima, Peru (2019). This research was performed in part to fulfill requirements for the MS degree from The University of Texas MD Anderson Cancer Center, UTHealth Graduate School of Biomedical Sciences; located within the University of Texas Medical Center in Houston, Texas 77030. We thank Gustavo Ayala, MD, for use of the Nuance Cri Multispectral Imaging System FX. This project was supported in part by NIH Grant 1R42-AI117990.
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5_2022_648_MOESM1_ESM.pptx
Supplementary file1 Fig. S.1 Lactoferrin treatment reduces pulmonary inflammation post infectious challenge with Mtb in a dose dependent response. Lungs from Mtb-infected mice were assessed at day 28 post aerosol infection (A) and compared to animals given bovine LF in the prophylactic group (B, C) or therapeutic group (D, E). Histologic assessment revealed primary granulomatous response with monocytic cell infiltration, dense cellular foci, and occluded vascular regions in control infected mice. Both prophylactic and therapeutic rHLF treatment reduced inflammatory response resulting in modest inflammatory foci and reduced pathological damage to lung tissue. While both doses (100 μg and 1 mg levels) were productive in limiting focal inflammation, the higher dose was more consistent between treatment groups. Hematoxylin and eosin-stained histographs represent formalin-fixed lung sections at 10× magnification obtained with 8–10 mice in each group; study representative of repeat experiments. Fig. S.2. Mycobacterial burden in lactoferrin-treated mice. C57Bl/6 mice were aerosol challenged with Mtb, strain Erdman, and treated with bovine lactoferrin (bLF) given as 100 μg or as 1 mg dose administered every other day orally beginning on day 14 (prophylactic treatment), or beginning on day 21 (therapeutic treatment) post infection. Lung (A), spleen (B) and liver (C) were removed on day 28 post infection; tissues were assessed for bacterial CFUs confirmed by plating serial dilutions on Middlebrook 7H11 agar plates using the large right lobe of the mouse lung that was weighed and homogenized into 2 mL PBS, which were subsequently incubated at 37°C for 3–4 weeks and represented as CFU burden per organ. Data are presented as individual mice with the mean and SEM indicated, n ≥ 6 mice per group. Fig. S.3. CellProfiler analysis of ofloxacin in histological sections. Example analysis is presented for pulmonary sections taken at 8 weeks post infection with MTB alone (A) or with lactoferrin treatment beginning at day 14 through day 28 (B). Each scanned section was assessed according to threshold parameters indicated. Counter-clockwise from top left shows (1) 100× histological scanned image; (2) single color outline of penetrating fluorescence; (3), color coding of ofloxacin signal detected (minus background), and (4) CellProfiler program threshold parameters Program parameters assume multiple objects in the image; colors indicate non-continuous regions of fluorescence highlighting separation of detected fluor to regions of intracellular accumulation (PPTX 1375 KB)
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Nguyen, T.K.T., Niaz, Z., Kruzel, M.L. et al. Recombinant Human Lactoferrin Reduces Inflammation and Increases Fluoroquinolone Penetration to Primary Granulomas During Mycobacterial Infection of C57Bl/6 Mice. Arch. Immunol. Ther. Exp. 70, 9 (2022). https://doi.org/10.1007/s00005-022-00648-7
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DOI: https://doi.org/10.1007/s00005-022-00648-7