Protocol for developing a mouse model of post-primary pulmonary tuberculosis after hematogenous spread in native lungs and lung implants

Summary

Here, we present a protocol for a mouse model for studying mechanisms of post-primary pulmonary tuberculosis (PTB) caused by virulent Mycobacterium tuberculosis (Mtb) using subcutaneous hock infection and lung tissue implantation. We describe steps for collagen instillation of lungs, lung and spleen implantation, preparation of Mtb for infection, and hock infection of mice. We then detail procedures for the perfusion of the lung and collection of organs, tissue processing, and histopathologic interpretation.

For complete details on the use and execution of this protocol, please refer to Yabaji et al.^1,2

Graphical abstract

Highlights

• •Selection of an Mtb-susceptible mouse genetic background
• •Guidance on dose and route of infection using virulent Mtb to induce lung TB lesions
• •Procedures for preparation of lung and spleen implants to study pulmonary TB lesions

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for a mouse model for studying mechanisms of post-primary pulmonary tuberculosis (PTB) caused by virulent Mycobacterium tuberculosis (Mtb) using subcutaneous hock infection and lung tissue implantation. We describe steps for collagen instillation of lungs, lung and spleen implantation, preparation of Mtb for infection, and hock infection of mice. We then detail procedures for the perfusion of the lung and collection of organs, tissue processing, and histopathologic interpretation.

Before you begin

This protocol describes a mouse model of pulmonary tuberculosis (PTB) using subcutaneous hock infection and lung tissue implantation in B6.Sst1S mice to study hematogenous dissemination, host-pathogen interactions, and genetic factors influencing disease progression.

Background

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading global health concern, causing significant morbidity and mortality. In human patients Mtb spreads hematogenously from primary infection sites and develops post-primary TB lesions in the lungs that lead to severe lung damage and Mtb spread via aerosols. Thus, one of the major challenges in combating TB is understanding mechanisms of TB progression in the lungs.

The development of animal models recapitulating key aspects of human disease is important for studying TB pathogenesis and evaluating novel vaccines and therapies. As in humans, lung is the most vulnerable organ targeted by virulent Mtb in experimental animals. However, modeling pulmonary TB (PTB) after hematogenous spread in immunocompetent hosts was unsuccessful.

In this protocol, we present a mouse model of post-primary PTB using an immunocompetent mouse strain B6.Sst1S mice genetically susceptible to Mtb. In this model mice are infected subcutaneously with virulent Mtb that spread to various organs, but PTB progression occurs specifically in the lungs and lung implants. This model recapitulates pathomorphological features of post-primary PTB lesions in humans, including granulomatous lesions with necrosis and fibronecrotic granulomas. This model allows to study lung-specific mechanisms of PTB progression, including interactions of immune and lung parenchymal cells.

To ensure that pulmonary TB lesions develop after initial priming of adaptive immunity and hematogenous spread, we infected B6.Sst1S mice with virulent Mtb suspension subcutaneously (SQ) in the hock, an established alternative to footpad injection.³ We have chosen the SQ hock infection to model the hematogenous TB spread, because 1) it allows clear separation of the primary site of infection from secondary, metastatic, lesions; 2) the only anatomically possible route of lung colonization is through hematogenous dissemination, resembling post-primary pulmonary TB progression in immunocompetent humans; 3) SQ hock injection is a common route of immunization,³ and T cell-mediated immunity is rapidly induced in the regional (popliteal) lymph node after subcutaneous and intradermal immunizations.^4,5

Institutional permissions

Readers need to obtain rights to perform animal experiments. For example, institutional permission to perform animal studies and collect tissues under an approved Institutional Animal Care and Use Committee (IACUC) or Institutional Review Board protocol. Our protocol was approved by Boston University’s Institutional Animal Care and Use Committee (IACUC protocol number PROTO201800218).

Mice

B6J.C3-Sst1C3HeB/Fej Krmn (B6.Sst1S, available from MMRRC stock # 043908-UNC). C57BL6/J (B6) and C3HeB/FeJ was purchased from Jackson Laboratory (Stock # 000658). (C3XB6.Sst1S)F1 hybrid was generated in our lab by crossing B6.Sst1S with C3HeB/FeJ.

These strains were selected because of their variable susceptibility to virulent Mtb. The B6 mice are relatively resistant to Mtb and do not develop necrotic pulmonary TB lesions. The B6.Sst1S, C3HeB/FeJ and their F1 hybrids are all homozygous for the sst1 susceptibility allels and develop progrressive pulmonary TB after aerosol infection, as described below. In our study, these strains were compared for their susceptibility to pulmonary TB after hematogenous spread from primary lesions, as described below.

Mouse selection and breeding

• 1.Use B6.Sst1S mice (B6J.C3-Sst1^C3HeB/FejKrmn) for this protocol, as this strain is genetically susceptible to TB due to the susceptible allele of the Sst1 locus, which promotes necrotic granulomatous lung inflammation.

Note: Maintain a colony of B6.Sst1S mice under specific pathogen-free (SPF) conditions to prevent confounding infections that may impact experimental outcomes.

Note: Consider the age of mice, as this can influence the immune response and disease progression. Typically, 8- to 12-week-old mice of both sexes can be used.

Preparation of donor lung or spleen tissue for implantation

CRITICAL: Lung or spleen tissue used for implantation must be obtained from pathogen-free donor mice. Ensure that the genetic background of the donor is appropriately matched for better acceptance of ingrafts.

• 2.Use B6 or B6.Sst1S mice as organ donor and B6.Sst1S mice as organ recipient.
• 3.Harvest lung and spleen tissue aseptically to avoid contamination. Use sterile instruments and appropriate biosafety procedures when handling lung tissue to maintain sterility and minimize the risk of external microbial influence.

Environmental and biosafety considerations

• 4.Perform all procedures involving live Mtb under BSL-3 conditions to ensure researcher safety and prevent environmental contamination.
• 5.Use personal protective equipment (PPE), including gloves, masks, and lab coats, and follow institutional biosafety protocols for handling infectious materials.

Animal handling, experimental controls, and reproducibility

• 6.Ensure appropriate analgesia and post-operative care for mice undergoing lung implantation surgery to reduce pain and distress.
• 7.Include appropriate controls, such as non-infected mice and mice without lung tissue implants, to account for baseline responses and procedural effects.
• 8.Standardize experimental conditions, including infection dose, time points for tissue collection, and environmental parameters, to minimize variability.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit Anti-Ttf1 monoclonal antibody, unconjugated, clone EP1584Y (1:100)	Abcam	Cat# ab76013, RRID:AB_1310784
Anti-Uteroglobin (Scgb1a1) antibody (EPR19846) (1:6,000)	Abcam	Cat# ab213203, RRID:AB_2650558
Iba1/AIF-1 (E4O4W) XP rabbit mAb (1:300)	Cell Signaling Technology	Cat# 17198, RRID:AB_2820254
Bacterial and virus strains
Mycobacterium tuberculosis Erdman	ATCC	N/A
Erdman(SSB-GFP, smyc′::mCherry)	6	N/A
Chemicals, peptides, antibiotics, and recombinant proteins
DMEM/Ham’s F-12 50/50 Mix [+] L-glutamine	Corning	Cat# 45000-344
Fetal bovine serum (FBS)	Hyclone	Cat# SH30071.03
10X Spectral DAPI	Akoya Biosciences	Cat# FP1490
Paraformaldehyde solution 4% in PBS	Fisher Scientific	Cat#
L-glutamine	Corning	Cat# 25-005-CI
Penicillin Streptomycin solution	Corning	Cat# 30-002-CI
HEPES buffer	Corning	Cat# 25-060-CI
L929 Cell conditioned media (LCCM)	This paper	N/A
Gibco 1X DPBS	Fisher Scientific	Cat#14-190-235
Middlebrook 7H9 Broth	BD Biosciences	Cat# 271310
Middlebrook 7H10 Agar	BD Biosciences	Cat# 262710
ProLong Gold Antifade Mountant	Invitrogen	Cat# P36934
Gibco Collagen I, rat tail	Fisher Scientific Company	A1048301
Ketamine	Zoetis Inc.	Cat#043-304
Xylazine	Dechra Vet Products, LLC	Cat#047-956
Heparin, sodium salt	Thermo Scientific	Cat#A1619803
2% Chlorhexidine in 70% alcohol	GAMA Healthcare Ltd.	Cat#CA2C240
Matrigel Basement Membrane Matrix	Corning	Cat#354234
Buprenorphine extended-release injectable suspension	Ethiqa XR, Fidelis Animal Health, Inc.	N/A
STERIS Vesphene III se Disinfectant cleaner	STERIS	Cat#647508
10 mL Control syringe	BD	Cat#309695
5 μm filter unit	Millex	Cat#SLSV025LS
Critical commercial assays
New Fuchsin method	Poly Scientific R&D Corp	Cat#K093
Experimental models: Organisms/strains
Mouse: C57BL/6J, adult male and female	The Jackson Laboratory	Stock No.: 000664
Mouse: B6J.C3-Sst1C3HeB/FejKrmn, adult male and female	Pichugin et al.7	Stock No: 043908-UNC https://www.mmrrc.org
Mouse: C3HeB/FeJ, adult male and female	The Jackson Laboratory	Stock No.: 000658
Mouse: (C3XB6.Sst1S)F1, adult male and female	This study	N/A
Mouse: B6.Sst1S;Ifnb-YFP, adult male and female	This study8,9	N/A
Software and algorithms
Graphpad Prism 9.5.1 (528)	GraphPad	N/A https://www.graphpad.com/
HALO v3.3.2541.262	Indica Labs, Inc. Clarivate Analytics Oxford Instruments Operatta Leica VT1200 S	https://indicalab.com/halo/ https://endnote.com/downloads https://imaris.oxinst.com/microscopy-imaging-software-free-trial? source=viewer https://www.perkinelmer.com/in/lab-solutions/product/operetta-cls-system-hh16000020 https://www.leicabiosystems.com/us/research/vibratomes/leica-vt1200/
EndnoteX9	N/A	N/A
Other
Tissue-Tek VIP-5 automated vacuum infiltration processor	Sakura Finetek	N/A
HistoCore Arcadia paraffin embedding machine	Leica	N/A
Automate in vivo manual gravity perfusion system for mice double 140 mL – IV 4140	Braintree Scientific, Inc.	Cat# IV 4140
Ultrospec 10 UV-visible spectrophotometer	Amersham Biosciences	Cat# 80-2116-30

Materials and equipment

Media 1

Reagent	Final concentration	Amount
DMEM/F12	–	430 mL
FBS	10%	50 mL
Penicillin-Streptomycin	1%	5 mL
1 M HEPES buffer	20 mM	10 mL
200 mM L-Glutamine	2 mM	5 mL

To prepare Media 1, add 10 mL of L929 Cell Conditioned Media (LCCM) to every 90 mL above media composition to a final concentration 10%.

Collagen I solution

Reagent	Final concentration	Amount
Collagen I (3 mg/mL)	1 mg/mL	335 μL
10X Media 1	–	100 μL
1N NaOH	–	17 μL
PBS	1X	548 μL

Note: Prepare freshly and keep it on ice.

Ketamine-xylazine solution (1 mL)

Reagent	Final concentration	Amount
Ketamine (100 mg/mL)	10 mg/mL	100 μL
Xylazine (20 mg/mL)	1 mg/mL	50 μL
PBS	1X	0.85 mL

Note: Prepare freshly and keep it at 22^oC–25^oC.

Heparin solution

Reagent	Final concentration	Amount
Heparin (100 IU/mg)	10 IU/mL	50 mg
1X PBS	1X	500 mL

Note: Prepare 10X sterile solution and store at 4°C for 1 month. Prepare 1X solution freshly.

Step-by-step method details

Timing: 2–4 h (for step 1)

Timing: 5–10 min per mouse (for step 1a)

Timing: 1–2 h (for step 1b)

Timing: 10–20 min (for step 1c)

Timing: 1–2 h (for step 1d)

Timing: 5–6 h (for step 2)

Timing: 3–4 h (for step 2a)

Timing: 1–2 h (for step 2b)

Timing: 5–7 days (for step 3)

Timing: 5–10 min per mouse (for step 3a)

Timing: 20 min per mouse (for step 3b)

Timing: 5 min per mouse (for step 3c)

Timing: 5 min per mouse (for step 3d)

Timing: 3–5 days (for step 3e)

• 1.Implant preparation.
  Here, we describe the steps for lung instillation with collagen I solution and lung and spleen implantation process.

  • a.
    Mouse lung instillation setup and preparation (Pre-instillation).

    • i.
      Anesthetize the mouse using ketamine-xylazine solution according to the weight of the mice (100 μl of ketamine-xylazine solution per 10g) intraperitoneally (i.p) or according to institutional animal care guidelines.
    • ii.
      Place the mouse on its back and secure it to a Styrofoam board with sterile pins to prevent movement during the procedure.
    • iii.
      Disinfect the fur on the mouse’s neck and chest with 70% ethanol to ensure a sterile field.
  • b.
    Collagen instillation of lungs.

    • i.
      Make a midline incision in the skin, starting a few millimeters above the urinary orifice and extending to the chin.
    • ii.
      Carefully separate the skin from the underlying muscle to expose the trachea.
    • iii.
      Rotate the Styrofoam board so that the trachea faces the operator.
    • iv.
      Insert an 18G catheter into the trachea slowly and gently to avoid tissue damage.
    • v.
      Remove the metal rod from the catheter once it is in place.
    • vi.
      Secure the catheter to the trachea using a piece of thread to prevent dislodgment.
    • vii.
      Load a 5 mL syringe with ice-cold collagen I solution (1 mg/mL).
    • viii.
      Inflate the lung by slowly injecting the collagen solution (around 1–1.5 mL) through the catheter.
    • ix.
      Use gentle, even pressure to avoid damaging the lung tissue.

      CRITICAL: Do not overinflate and avoid lung rupture.

    • x.
      After inflation, carefully remove the catheter.
    • xi.
      Tie off the trachea securely to prevent any fluid leakage.
    • xii.
      Excise the trachea along with the entire inflated lung using sterile dissection tools.

      Note: Lung removal was performed under ketamine-xylazine anesthesia, which results in the death of the animal and does not require an additional euthanasia step.

  • c.
    Incubation of collagen instilled lungs.

    • i.
      Incubate the lung at room temperature submerged in sterile DMEM F12 media containing 10% FBS and antibiotic.
    • ii.
      Incubate for 10 minutes to allow the collagen to solidify and stabilize the lung architecture.
    • iii.
      Trim the lung approximately 10 mm in size using sterile scissors, selecting the left lobe or a suitable right lobe segment.
    • iv.
      Get the spleen from same mouse and also make the similar size tissue after trimming with sterile scissors.
    • v.
      Place the prepared tissue grafts into sterile tissue culture medium containing antibiotics to prevent contamination.

      Note: Ensure all tools and surfaces are sterile to avoid contamination, and handle the lung tissue gently to preserve its structural integrity.

  • d.
    Lung and spleen implantation.

    • i.
      Anesthetize recipient mice (6 weeks of age or older) with isoflurane and place them in a sternal recumbency position.
    • ii.
      Prepare the surgical site by shaving the area between the scapulae.
    • iii.
      Clean the shaved area with 2% chlorhexidine followed by 70% ethanol, repeating the cleaning process three times to ensure sterility.
    • iv.
      Make a midline incision of approximately 1–2 cm through the skin between the scapulae using sterile surgical tools.
    • v.
      Create subcutaneous pockets on both sides of the incision by blunt dissection.
    • vi.
      Remove the lung and spleen grafts from the antibiotic-containing culture medium.
    • vii.
      Dip each graft in Corning Matrigel Basement Membrane Matrix, LDEV-free (Corning, NY, USA; product number 354234) to coat the tissue.

      CRITICAL: It is important to pre-coat the tissues with Corning Matrigel Basement Membrane Matrix before implantation to enhance cell survival, adhesion, and engraftment by providing an extracellular matrix (ECM)-like environment.

    • viii.
      Insert the Matrigel-coated lung graft into one subcutaneous pocket.
    • ix.
      Place the spleen graft (or additional lung graft) into the contralateral subcutaneous pocket.
    • x.
      Close the incision using absorbable sutures and secure the skin with skin glue.
    • xi.
      Administer analgesic treatment with extended-release buprenorphine (Ethiqa XR, Fidelis Pharmaceuticals LLC) subcutaneously at the time of surgery.
    • xii.
      Allow the mice to recover from anesthesia under close observation.
    • xiii.
      Once recovered, return the mice to their colony housing with their original cage-mates.
    • xiv.
      Monitor the mice post-operatively for any signs of pain or distress, providing additional care as needed.
    • xv.
      Confirm the acceptance of grafts after 2 months post implantation by manual and visual inspection.

      Note: Perform the lung instillation and organ implantation on same day.

• 2.Mtb infection.
  Here, we describe the steps for preparing Mycobacterium tuberculosis (Mtb) cultures for mouse infection and performing the infection.

  • a.
    Preparation of Mtb for infection.
    The detailed protocol for this step is provided in¹⁰ and briefly explained below.

    • i.
      Perform all steps in a Biosafety Level 3 (BSL3) facility using approved protocols and disinfectants inside a Biosafety Cabinet (BSC).
    • ii.
      Dilute 0.5–1.0 mL of frozen Mtb Erdman (SSB-GFP, smyc′::mCherry) stock in 5 mL Middlebrook 7H9 Broth with OADC and transfer to a sterile media bottle.
    • iii.
      Place the bottle in a secondary container with absorbent paper and a leak-proof lid.
    • iv.
      Incubate at 37°C in a shaking incubator at 100 rpm for 2–3 days until OD600 is 0.4–0.5.
    • v.
      Transfer the Mtb suspension to a sterile 15 mL conical tube and centrifuge at 2100×g (∼3000 rpm) for 10 minutes at 20°C–25°C.
    • vi.
      Resuspend the pellet in 5 mL pre-warmed 1X PBS.
    • vii.
      Sonicate twice for 5 seconds each with a 5–10 second interval in a sonicating water bath.
    • viii.
      Add 5 mL more pre-warmed 1X PBS, mix, and let sit vertically for 30–60 minutes.
    • ix.
      Use the upper 8 mL without disturbing the clumps at the bottom.
    • x.
      Attach a 5 μm filter to a syringe barrel, place over a conical tube, and transfer the suspension.
    • xi.
      Insert the plunger and press gently to filter, avoiding gravity filtration to prevent Mtb loss.
    • xii.
      Measure OD600 of the filtered suspension; expect 0.1–0.2, assuming OD600 = 1 equals ∼3×10⁸ bacteria/mL of the single cell suspension of Mtb obtained after filtration.
    • xiii.
      Dilute the Mtb suspension with 1X PBS containing 10⁶ Mtb per 50 μL for mice infection.
    • xiv.
      Plate an aliquot on solid media (MB7H10) and count colonies after 3 weeks to confirm the infectious dose.
  • b.
    Infection of mice.

    • i.
      Anesthetize the B6.Sst1S mice containing implants as described in step 1a.
    • ii.
      Confirm that the mice are fully anesthetized before proceeding to ensure no movement or discomfort.
    • iii.
      Place the anesthetized mouse in a restrainer with access to the hind limb.
    • iv.
      Identify the lateral tarsal region just above the ankle (hock) of the mouse.
    • v.
      Using a sterile 28G needle and 1 mL syringe, inject 50 μL of the bacterial suspension (prepared in step 2a xiii) subcutaneously into the lateral tarsal region of the hind limb.

      Note: Formation of a bubble is a sign of proper subcutaneous delivery.

    • vi.
      Monitor the mouse for proper recovery from anesthesia, ensuring it is returned safely to its cage with minimal stress.
• 3.Collection and processing of infected organs for imaging.
  Here, we describe the steps for lung perfusion and collection of organs from Mtb infected mice for downstream analyses.

  • a.
    Mouse perfusion setup and preparation (Pre-perfusion).

    • i.
      At the desired time points post infection (12–24 weeks post infection), anesthetize the mice described in step 1a.
    • ii.
      To prevent blood coagulation, inject 200 μl of 1X PBS containing 100 IU/ml heparin intraperitoneally (i.p.).
    • iii.
      Set up the Automate in vivo manual gravity perfusion system: fill one syringe with 20 ml of 4% PFA in PBS and another syringe with 80 ml of PBS/heparin solution (10 U/ml). Attach a 20G needle.
    • iv.
      Pin the mouse securely to a Styrofoam board to immobilize it.
    • v.
      Disinfect the fur thoroughly with 70% ethanol using a squeeze bottle.
  • b.
    Mouse lung perfusion procedure (PBS + Heparin followed by 4% PFA).

    • i.
      Make a midline incision from just above the urinary orifice up to the chin.
    • ii.
      Separate the skin from the underlying muscle layer to expose the abdominal area and cut peritoneum to expose intestine.
    • iii.
      Do not open the rib cage to prevent lung collapse.
    • iv.
      Gently move the intestines to the left side to expose the inferior vena cava, ensuring no organs obstruct blood flow during perfusion.
    • v.
      Cut the skin near the lower limbs to facilitate blood drainage, maintaining continuous flow to prevent coagulation (avoid a “blood swamp”).
    • vi.
      Position the dissection board upright within the tray.Make small incisions to cut the inferior vena cava and aorta in abdominal area.
    • vii.
      Rapidly insert a 20G needle attached to the Automate in vivo manual gravity perfusion system into the retro-orbital sinus, adjusting as needed, and begin perfusion with PBS + Heparin using the perfusion assembly.
    • viii.
      Observe pulse-like blood flow, perfuse until blood is replaced by clear PBS. Use approximately 20 mL of PBS + Heparin mix.
    • ix.
      Switch to 4% PFA and continue perfusing with about 10 mL of fixative.

      Note: Before starting the procedure of next mouse flush the system with 5 mL of PBS + Heparin to remove any PFA residues.

    • x.
      Lower the dissection board and place tools in a Falcon tube filled with 1% Vesphene.
  • c.
    Lung inflation and bronchoalveolar lavage procedure.

    • i.
      Hold the sternum with tweezers and make a small hole in the diaphragm to collapse the lungs. Carefully cut the rib cage, being cautious not to cut the lung.
    • ii.
      Extend cuts along the diaphragm to fully open the thoracic cavity.
    • iii.
      Make a midline incision from the sternum to the chin, spread the rib cage, and pin it open, avoiding sharp parts.
    • iv.
      Rotate the board so the trachea faces the operator. Place a thread beneath the trachea.
    • v.
      Slowly insert an 18G catheter into the trachea without force. Remove the metal rod and place it in 1% Vesphene. Tie the catheter in place.
    • vi.
      Attach an insulin syringe with 1 mL of PBS and inflate the lungs.
    • vii.
      Collect bronchoalveolar lavage (BAL) fluid if required.
    • viii.
      Repeat inflation 3–5 times to remove residual air.

      CRITICAL: Do not overinflate and avoid lung rupture.

    • ix.
      Inflate the lungs slowly with 2 mL of PBS. Remove the catheter and immediately tie off the trachea.
  • d.
    Tissue collection and fixation procedure.

    • i.
      Isolate the lungs and place them in a 50 mL Falcon tube containing 10% formalin. Flip the tube upside down to ensure complete submersion.
    • ii.
      Collect other organs (spleen, liver, kidney, gut, lymph nodes, and any others required for the study) and place them in a 50 mL Falcon tube filled with 10% formalin. Secure the tube properly.
    • iii.
      Remove pins, reverse the mouse to face down, and pin the limbs.
    • iv.
      Open the back skin using scissors and forceps to collect any implanted tissues. Place them in 10% formalin.
    • v.
      Place all dissection tools in 1% Vesphene solution and clean the biosafety cabinet thoroughly.
    • vi.
      Fix tissues for 24–48 hours. After fixation, remove them from BSL-3 containment and transfer to sterile 1X PBS in a 50 mL Falcon tube.
  • e.
    Tissue processing, and histopathologic interpretation.

    • i.
      Perform the tissue processing and histopathological interpretation using standard classical methods.
    • ii.
      Stain a subset of tissue section for acid fast bacteria (AFB).
    • iii.
      For bright-field acid-fast staining of Mtb-infected lung sections, use the New Fuchsin method (Poly Scientific R&D Corp., Cat. No. K093, Bay Shore, NY, USA). https://www.statlab.com/pdfs/ifu/IFU_KTAFB_Acid_Fast_Bacteria_(A.F.B.)_Stain_Kit_Procedure_01152019.pdf

      Alternatives: You can use other methods of Mtb staining like fluorescent Acid-Fast Bacteria (AFB) Staining using Auramine-Rhodamine Method.

    • iv.
      If required the lungs can be processed for 3D imaging using thick sections.¹¹

Expected outcomes

I. Progression of PTB in native lungs of B6.Sst1S. In pilot studies, we infected adult female mice via subcutaneous hock injection with 10⁴, 10⁵ and 10⁶ CFU of Mtb Erdman and only 10⁶ CFU induced pulmonary granulomas in 100% of animals by 12 weeks post-infection (wpi) (Table 1). However, when we infected the wild type B6 and congenic B6.Sst1S mice with 10⁶ CFU and observed the weight loss and decreased survival only in the B6.Sst1S mice.^1,12 Groups of B6 and surviving B6.Sst1S mice were sacrificed at 11- and 20-weeks post-infection (wpi), developing PTB lesions in both strains. Based on Mtb load and histopathological features in the pulmonary lesions we categorized the lesions in two types, classified as “paucibacillary” and “multibacillary”. Majority of B6.Sst1S develop multibacillary pulmonary lesions while B6 mice only develop paucibacillary pulmonary lesions (Table 2). B6 mice develops only minimal to mild granulomatous interstitial lesions containing rare single acid-fast bacilli-AFB, while B6.Sst1S lesions displayed a broad variability of lesions including severe lesions characterized by the presence of cholesterol clefts, neutrophils and areas of necrosis with innumerable AFB^1,12 (Figure 1). Moreover, we compared the males and females B6.Sst1S mice for Mtb infection and similar disease progression was observed in both sexes (Table 3). These findings emphasize the role of Sst1 locus in determining pulmonary TB severity and survival. II. Effect of genetic background and age on TB progression in native lungs. We further investigated the genetic influence on tuberculosis (TB) progression in different sst1-susceptible mice, using B6.Sst1S, C3HeB/FeJ (HeB), and F1 hybrids (HeB x B6.Sst1S). We infected all strains with Mtb at 10⁶ CFU subcutaneously. By 14–20 all strains developed TB lesions; however, B6.Sst1S mice displayed more multibacillary lesions with necrosis compared to HeB and F1 hybrids (Table 4). We also observed survival differences and B6.Sst1S mice showed 67% survival with weight loss, whereas HeB and F1 hybrids demonstrated 100% survival rates and no significant weight loss throughout the course of infection (Figures 2A and 2B). We also measured Mtb loads in several organs and observed the highest bacterial burden in the lungs of B6.Sst1S mice, with bacterial dissemination to secondary organs, including the spleen, liver, lymph nodes, and gut (Figure 2C). In the extrapulmonary organs Mtb is rarely present and localized exclusively within small macrophage aggregates. Additionally, to confirm the effect of age on disease progression, we selected the F1 hybrid strain with the least disease severity and infected young and adult (HeB × B6.Sst1S)F1 mice with Mtb via the subcutaneous route using 10⁶ CFU. Interestingly, younger F1 mice exhibited more multibacillary lesions compared to their adult counterpart (Table 5). These findings emphasize the critical role of genetic background in determining pulmonary TB severity and survival. Host genetics significantly impact lesion control, bacterial load, and disease progression. III. Investigating TB progression in lung and spleen implants. We investigated the progression of PTB lesions in the native lungs after hematogenous spread under pre-existing condition. Further we wanted to study lung-specific TB progression using subcutaneous implantation of lung tissue fragments from B6.Sst1S into B6.Sst1S mice to study the effect of oxygenation and lung microenvironment. We co-implanted spleen fragments as controls. Additionally, we compared the lung implants with 1% collagen instillation or without instillation before implantation. Two months post-implantation, mice were infected via the subcutaneous hock route, and tissues were collected, and we observed a better preservation of structural integrity of tissue in collagen instilled condition (Figure 3). We observed the formation of organized necrotic granulomas and fibrotic capsules with innumerable Mtb in implanted lung, while spleen implants rarely show bacteria without granulomas (Figures 4A–4C). Using fmIHC, we confirmed the presence of lung specific markers in the lung implants including epithelial markers Nkx2.1 (Ttf1), uteroglobin and we also included macrophage marker, Iba1. The lung specific markers were exclusively present in the implanted lung but not in the implanted spleen. However, Iba1 was expressed in both implanted tissues (Figure 4D). We also observed a correlation between the native and implanted lungs. Paucibacillary lesions in the native lung were associated with an absence of lesions and Mtb in the implanted lungs. In contrast, multibacillary lesions in the native lungs led to lesion formation and Mtb replication in the implanted lungs (Figures 5A and 5B). To investigate the effect of the recipient mice’s genetic background on implanted lung tissue from wild-type B6 mice, we subcutaneously implanted lung tissue from wild-type B6 mice into B6.Sst1S mice. After recovery, these mice were infected with 10⁶ CFU of Mtb Erdman. At 20 weeks post-infection, the implanted B6 lung also developed necrotic lesions with numerous Mtb (Figure 5C).

Table 1.

Dose-dependent progression of Mtb infection in B6.Sst1S mice

Mtb dose	Time post infection(Weeks)	Lung	Spleen	Lymph nodes	Percentage of mice forming pulmonary granulomatous lesions
104	12	No lesions or sign of Mtb	No lesions or sign of Mtb	No lesions or sign of Mtb	(0/5) 0%
105	12	Very small lesions	Few Mtb with no progression of disease	Few Mtb with no progression of disease	(3/6) 50%
106	12	Small-medium size lesions associated with Mtb	Few Mtb with no progression of disease	Few Mtb with no progression of disease	(5/5) 100%

Table 2.

Effect of genetic background on PTB lesion progression in mice within 11–20 weeks post-infection

Genotypes	Sex	Multibacillary	Paucibacillary	No lesions	Total
B6	Female	0 (0%)	7 (77.7%)	2 (22.23%)	9
B6.Sst1S	Female	24 (92%)	2 (8%)	0 (0%)	26

Figure 1.

Progression of PTB lesions in native lungs of B6.Sst1S

Stages of PTB progression in B6.Sst1S mice following subcutaneous Mtb infection. Representative low-magnification and corresponding high magnification (200X) histopathology images of lung sections (H&E staining). Insets in low magnification images showing the area selected for high magnification images. The panels depict three categories of lesions: paucibacillary (n = 5), multibacillary intermediate (left panel, n = 10), and multibacillary advanced (right panel, n = 10). The intermediate type lesions are characterized by single-cell necrosis with intracellular bacterial replication, seen as clumps. The advanced lesions display severe lung damage with necrosuppurative pneumonia containing extracellular clumps of Mtb. Representative images from two different mice are used to illustrate these lesion types. Scale bar = 1.5 mm (top panels) and 200 μm (bottom panels).

Table 3.

Effect of sex on PTB lesion progression

Genotypes	Sex	Multibacillary	Paucibacillary	No lesions	Total
B6.Sst1S	Male	6 (75%)	2 (25%)	0	8
B6.Sst1S	Female	9 (64.3%)	3 (21.4%)	2 (14.3%)	14

Within 24 weeks post infection.

Table 4.

Effect of genetic background on PTB lesion progression in sst1-susceptible mice within 12–20 weeks post-infection

Genotypes	Multibacillary	Paucibacillary	No lesions	Necrosis	Total	Sex
B6.Sst1S	24 (92%)	2 (8%)	0	Yes	26	Female
(C3HXB6.Sst1S)F1	2 (10.5%)	14 (73.7%)	3 (15.8%)	No	19	Female
C3HeB/FeJ	4 (33.3%)	8 (66.7%)	0	No	12	Female

Figure 2.

Comparison of post-primary PTB progression in sst1-susceptible B6.Sst1S, C3HeB/FeJ mice and their F1 hybrids

(A) Survival curves of mice infected with M. tuberculosis. Survival of C3HeB/FeJ (n=12), F1 (FeJ∗1H) (n=23) and B6.Sst1S (n=37) following hock infection with 10⁶ CFU of M. tuberculosis Erdman(SSB-GFP, smyc′::mCherry). The Log-rank (Mantel-Cox) test was applied to determine the statistical significance.

(B) Weight of the infected mice: C3HeB/FeJ (n=12), F1 (FeJ∗1H) (n=23) and B6.Sst1S (n=37) following hock infection with 10⁶ CFU of M. tuberculosis Erdman(SSB-GFP, smyc′::mCherry). The statistical significance was performed by two-way ANOVA using Tukey’s multiple comparison test.

(C) Semi-quantitative acid-fast bacteria (Mtb) loads in the lungs, spleens , popliteal lymph nodes and livers of the infected mice at 14 weeks post infection. Each hash on the X-axis represents individual mouse. Organs are shown in different colors. The p value ≤0.05 was considered statistically significant. Significant differences are indicated with asterisks (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001).

Table 5.

Effect of age on PTB lesion progression in sst1-susceptible mice within 12–20 weeks post-infection

Genotypes	Age	Multibacillary	Paucibacillary	No lesions	Total	Sex
(C3HXB6.Sst1S)F1	Adult	2 (10.5%)	14 (73.7%)	3 (15.8%)	19	Female
(C3HXB6.Sst1S)F1	Young	4 (57.1%)	2 (28.6%)	1 (14.3%)	7	Female

Figure 3.

Effect of collagen I instillation on lung implant condition

H&E-stained images at low and high magnification of implanted lungs from B6.Sst1S mice with or without collagen instillation. 1% Collagen I solution in tissue culture medium was instilled prior to implantation into recipient mice. B6.Sst1S mice were hock infected with 10⁶ CFU of Mtb and sacrificed at 20 wpi. Insets in low magnification images showing the area selected for high magnification images. For high-magnification images, the scale bar represents 1 mm in the top panels, 200 μm for the 100X magnification images, and 50 μm for the 400X magnification images.

Figure 4.

TB lesions in lung implants

(A) Low and high magnification images showing H& E and AFB staining of implanted lung and spleen. Scale bar = 1 mm.

(B) The Mtb and Mtb induced lesions were only present in implanted lung but not in implanted spleen. For high-magnification images, the scale bar represents 100 μm for the top panels (200X) and 50 μm for the bottom panels (400X).

(C) Masson’s Trichrome staining of implanted lung showing fibrotic capsule surrounding necrotic granuloma in a lung implant. 100X magnification. Scale bar = 200 μm.

(D) Fluorescent multiplexed immunohistochemistry (fmIHC) was performed on native lungs, as well as lung and spleen implants, from Mtb-infected B6.Sst1S mice. Tissue sections were stained for lung epithelial cell markers Ttf1 (Nkx2.1) and Uteroglobin (Scgb1a1), along with the macrophage marker Iba1. Scale bar represents 200 μm (50X), 100 μm (100X), and 50 μm (200X), respectively. Yellow inset boxes in 50X- and 100X-magnification images indicate the regions selected for corresponding higher-magnification images. White inset box in lung implant image at 50X denotes the area presented at 200X magnification.

Figure 5.

Correlation between disease progression in native lungs and implanted lungs

(A) H&E and AFB images of pauci- and multibacillary TB lesions in native lungs. Insets in low magnification images showing the area selected for high magnification images. For high-magnification images, the scale bar represents 1.5 mm (left panels), 200 μm for the middle panels (100X) and 50 μm for the right panels (400X).

(B) AFB staining of the corresponding lung and spleen implants isolated from the same mice presented in A. Multibacillary TB lesions were found only in the lung implants of mice with multibacillary lesions in their native lungs. Scale bar = 50 μm.

(C) H&E and AFB images showing TB lesions in the native lung of B6.Sst1S recipient mice (upper panel) and in their implanted lung tissue isolated from B6 donors mice (lower panel). Implanted B6 lung tissue developed the necrotic lesions containing numerous Mtb. Inset in low magnification image showing the area selected for high magnification image. Scale bar represents 1.5 mm (1X), 200 μm (100X), 100 μm (200X), and 50 μm (400X).

Quantification and statistical analysis

To compare multiple groups with two or more variables, we conducted a two-way analysis of variance (ANOVA) and applied adjustments for multiple post hoc comparisons. Various comparisons were made, including comparisons among all groups, between the control group and all other groups, and between selected groups. For comparisons involving multiple groups with a single variable, a one-way ANOVA was used, with corrections for multiple post hoc comparisons. In cases where only two groups were compared, two-tailed paired or unpaired t-tests were employed. The Log-rank (Mantel-Cox) test was utilized to assess statistical significance in mouse survival data. All statistical analyses were performed using GraphPad Prism 9 software. A p-value of < 0.05 was considered statistically significant, and statistical significance was indicated by asterisks (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001).

Limitations

The subcutaneous infection model does not recapitulate the natural route of infection in the majority of humans. However, it allows for clear separation between the primary and post-primary “metastatic” pulmonary TB lesions and demonstrates lung vulnerability to Mtb irrespective of the route of infection. The lung implantation model complements this by enabling the study of the lung tissue-specific elements promoting TB progression, although it cannot fully replicate the native lung’s architecture, vascularization, oxygenation, and ventilation potentially impacting TB progression. Implant stability is influenced by tissue size and preparation, as smaller fragments are often resorbed. Histocompatibility of the host and lung implant need to be considered.

Troubleshooting

Problem 1

Appropriate dose of Mtb to establish a reliable and reproducible subcutaneous infection in mice, ensuring consistent infection dynamics while minimizing variability and overexposure.

Potential solution

Use the optimum dose (10⁶) of Mtb Erdman to consistently induce pulmonary granulomatous lesions in 100% of mice by 12 weeks post-infection (wpi) (Step 2a xiii, Table 1).

Problem 2

Selection of mice background to get pulmonary necrotic granulomatous lesions by subcutaneous infection.

Potential solution

Use B6.Sst1S mice (B6J.C3-Sst1^C3HeB/FejKrmn) to induce pulmonary necrotic granulomatous lesions through subcutaneous infection in the hock. Monitor lesion progression, which drives Mtb replication. Use of B6, C3H, or F1 mice results into formation of pulmonary lesions with reduced bacterial loads and tissue damage; however, these lesions do not progress to necrotic granulomatous lesions (Table 4).

Problem 3

Disappearance or collapse of smaller implanted tissues from recipient mice.

Potential solution

Observed that smaller tissue sections disappear during recovery and vascularization, failing to establish stable implants. Use about half of the left lobe or one of the right lobes to implant which remain stable and persist throughout recovery and critically determines the success of lung implantation (Step 1b).

Problem 4

How can structural integrity of lung tissue be maintained during implantation to improve implantation success?

Potential solution

Instill lungs with 1% collagen before implantation to support and preserve tissue structure. Collagen-instilled lung implants show significantly better structural integrity compared to untreated controls, demonstrating that collagen instillation is essential for maintaining lung architecture and enhancing implantation outcomes (Step 1b, Figure 3).

Problem 5

Variation in PTB progression in individual mice after hock infection.

Potential solution

To detect organized granulomas in lung implants, monitor weight and clinical scores weekly and sacrifice mice when they start losing weight, typically around 12–24 weeks post-infection. This timing increases the likelihood of obtaining lung implants with lesions and detectable Mtb (Step 3, Figure 5A). Implant spleen or other non-lung tissues to verify the specificity of lung susceptibility to TB infection following hematogenous spread.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Igor Kramnik (ikramnik@bu.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by Shivraj M. Yabaji (smyabaji@bu.edu).

Materials availability

All unique/stable reagents generated in this study are available from the lead contact with a completed materials transfer agreement.

Data and code availability

This study did not generate datasets/code, and any additional information will be available from the lead contact upon request.

Acknowledgments

The work was supported by NIH R01HL126066 (I.K.) and NIH grants S10OD026983 and S10OD030269 (N.A.C.).

Author contributions

Conceptualization, I.K. and S.M.Y.; methodology, S.M.Y., M.L., S.L., I.G., A.K.O., H.P.G., and C.E.T.; validation and formal analysis, S.M.Y., M.L., and N.A.C.; investigation, S.M.Y., S.L., I.G., L.K., and N.A.C.; resources, N.A.C. and I.K.; writing – original draft, S.M.Y.; writing – review and editing, I.K., L.K., and N.A.C.; supervision, I.K.; funding acquisition, I.K., N.A.C., and L.K.

Declaration of interests

The authors declare no competing interests.