Tumor necrosis factor neutralization results in disseminated disease in acute and latent Mycobacterium tuberculosis infection with normal granuloma structure in a cynomolgus macaque model

Abstract

Objective: An increased risk of tuberculosis has been documented in humans treated with tumor necrosis factor alpha (TNFalpha)-neutralizing agents. In murine models, impaired signaling by TNF causes exacerbation of both acute and chronic infection associated with aberrant granuloma formation and maintenance. This study was undertaken to investigate immune modulation in the setting of TNF neutralization in primary and latent tuberculosis in a non-human primate model.

Methods: Cynomolgus macaques 4 years of age or older were infected with Mycobacterium tuberculosis and subjected to clinical, microbiologic, immunologic, and radiographic examinations. Monkeys were classified as having active or latent disease 6-8 months after infection, based on clinical criteria. Monkeys used in acute infection studies were randomized to receive either adalimumab (prior to and during infection) or no treatment. Monkeys with latent infection that were randomized to receive TNF-neutralizing agent were given either an inhibitor of soluble TNF, recombinant methionyl human soluble TNF receptor I (p55-TNFRI), or adalimumab. Control monkeys with latent infection were given no treatment or saline. Data from previously studied monkeys with active or latent disease were also used for comparison.

Results: Administration of TNF-neutralizing agents prior to M tuberculosis infection resulted in fulminant and disseminated disease by 8 weeks after infection. Neutralization of TNF in latently infected cynomolgus macaques caused reactivation in a majority of animals as determined by gross pathologic examination and bacterial burden. A spectrum of dissemination was noted, including extrapulmonary disease. Surprisingly, monkeys that developed primary and reactivation tuberculosis after TNF neutralization had similar granuloma structure and composition to that of control monkeys with active disease. TNF neutralization was associated with increased levels of interleukin-12, decreased levels of CCL4, increased chemokine receptor expression, and reduced mycobacteria-induced interferon-gamma production in blood but not in the affected mediastinal lymph nodes. Finally, the first signs of reactivation often occurred in thoracic lymph nodes.

Conclusion: These findings have important clinical implications for determining the mechanism of TNF neutralization-related tuberculosis.

Figure 1

TNF is necessary to control initial and latent M. tuberculosis infection. A) Gross disease pathology in macaques infected with low dose M. tuberculosis with or without TNF neutralizing antibody for 8 weeks. Control monkeys show granulomas (black circles) localized to a single lung lobe and mediastinal lymph node enlargement (grey circles) with or without granulomas (checkered circles). In 3 of 4 monkeys treated with anti-TNF reagent, aggressive and disseminated disease was observed including multiple granulomas in lungs, liver and spleen and tuberculous pneumonia (checkered areas in the lungs). B) Latently infected monkeys have limited gross pathology: a few granulomas in the lung, and mediastinal lymph node enlargement with or without granulomas. In contrast, gross pathology of latent monkeys treated with TNF neutralizing agents ranged from normal to severe disseminated disease. Liver and spleen (monkeys 12102, 23802, 25503, 6604 and 7104) had multiple granulomas to miliary disease (speckled pattern). Disseminated miliary disease was observed in all lung lobes, liver and spleen of 23802. Multiple granulomas were observed on the diaphragm (dotted line) of 6604 and extensive pleural disease (dotted line) in 25503. Individual monkey identifiers are below each schema with necropsy score in parentheses.

Figure 2

Gross pathology and bacterial burden of acute and latent control and TNF neutralized monkeys. (A) During acute infection, gross pathology as quantified by necropsy score appeared greater among TNF neutralized (open circles) compared to control monkeys (open squares) though this did not reach statistical significance. Overall bacterial burden (CFU score) and distribution of bacterial burden (percent of positive samples) appeared higher in anti-TNF treated monkeys, but not statistically different due to variability in the groups. (B) Latently infected monkeys treated with TNF neutralization agents (closed circles) results in greater gross pathology, bacterial burden, and dissemination (percent positive samples) compared to latent control monkeys (closed squares) (Mann Whitney or Student’s T test). Latently infected monkeys treated with adalimumab are indicated by solid circles whereas monkeys treated with p55-TNFR1 are indicated in solid stars. (* is p<0.05, ** is p<0.01).

Figure 3

More aggressive disease is seen in TNF neutralized monkeys with normal granuloma structure. (A) Caseous granuloma with adjacent non-necrotizing granulomas (left side) present in the hilar lymph node of an 8 week control monkey (18007), 5×; (B) Caseous granuloma in the accessory lobe of acute control monkey (9703) consisting of a central area of eosinophilic proteinaceous material (caseum) surrounded by epithelioid macrophages and the lymphocytes along the outmost periphery, 5×; (C) Massive effacement of carinal lymph node with both caseous and non-necrotizing granulomas in a TNF neutralized monkey 8 weeks after infection, 2×; (D) Large caseous granuloma in the lung of TNF neutralized monkey (2807) with normal architecture, 2×; (E) Large caseous granuloma in the lung lobe of a monkey with active tuberculosis. The architectural structure of the granuloma appears similar to granulomas observed in monkeys treated with TNF neutralizing agents, 2×. All slides stained with H&E.

Figure 4

TNF neutralization alters cytokine and chemokine receptor expression during acute and latent infection. (A) Lung and lymph node homogenates were analyzed for cytokine production. During acute infection, TNF neutralization (open circles) resulted in more IL-2 in lungs and less IL-8 in lung granuloma compared to control groups (open squares). Increased IL-12p70 was observed in lung and hilar lymph nodes among latently infected monkeys treated with TNF neutralizing agents (closed circles) compared to active (closed squares) and latent (closed triangles) controls. CCL4 was reduced in TNF neutralized groups compared to active and latent controls. (Kruskal-Wallis with Dunn’s multiple comparison). (B) Flow cytometry dot plots of CD4 T cell gate and CXCR3+CCR5+ dual positive cells in PBMC and affected lung samples. Significantly greater percentages of CXCR3+CCR5+ CD4 and CD8 T cells are observed among TNF neutralized monkeys compared to active and latent groups. (ANOVA with Bonferroni post hoc analysis.) * p<0.05, ** p<0.001

Figure 5

Normal granuloma structure after TNF neutralization despite reactivation tuberculosis. Latently infected monkeys have caseous and/or mineralized granulomas in lungs and mediastinal lymph nodes. (A) Mineralized granuloma (central core of mineral has artifactually dropped out of section) surrounded by a fibrotic rim within a mediastinal lymph node, 5×; (B) Focal mineralized granulomas (mineral shattered during cutting) in lung of latent monkey, 5×; (C) Non-necrotizing granulomas (arrow) emanating from caseous granuloma (left) with mineralization suggesting reactivation is occurring from carinal lymph node of this natural reactivation monkey, 2×; (D) Classic caseous granuloma in lung with a proteinaceous central area of necrosis surrounded by epithelioid macrophages and lymphocytes in a natural reactivation monkey, 5×; (E) Mineralized granulomas (and caseous granulomas) in carinal lymph node of a TNF neutralized monkey. (F) Caseous granuoloma with fibrocalcific changes in TNF neutralized monkey, indistinguishable from a latent control granuloma, 5×; (G) Non-necrotizing granulomas (arrow) emerging from a caseous granulomas (with mineralization) in TNF neutralized monkey suggesting reactivation from carinal lymph node, similar to (C), 2×; (H) Classic caseous granuloma structure in lung of 25503 who had disseminated disease throughout the lung, liver and spleen.5×. All slides stained with H&E.