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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2013 Apr 4;110(16):6482–6487. doi: 10.1073/pnas.1219985110

CD4+ cell-dependent granuloma formation in humanized mice infected with mycobacteria

Frank Heuts a, Dolores Gavier-Widén b, Berit Carow a, Julius Juarez c, Hans Wigzell a, Martin E Rottenberg a,1
PMCID: PMC3631626  PMID: 23559373

Abstract

We have used humanized mice, in which human immune cells differentiate de novo from transplanted cord blood progenitor cells, to study the human immune responses to infection with Mycobacterium bovis bacillus Calmette–Guérin and Mycobacterium tuberculosis. Granulomas with a core containing giant cells, human CD68+ macrophages, and high bacilli numbers surrounded by a layer of CD3+ T cells and a fibrotic response encapsulating the lesions were observed in livers and lungs from bacillus Calmette–Guérin-infected humanized mice but not in nonhumanized infected controls. Paradoxically, humanized mice contained higher mycobacterial numbers in organs than nonhumanized controls. The enhancement of bacterial load was mediated by human CD4+ cells and associated to an increased expression of Programmed Death-1 protein and CD57 on T cells, molecules associated with inhibition and senescence. The lesions from mice depleted of CD4+ cells were scarcer, minimal, and irregular compared with those from mice depleted of CD8+ cells or nondepleted controls. Granulomas of bacillus Calmette–Guérin-infected humanized mice administered with a TNF-neutralizing TNF receptor fusion molecule preserved their structure, but contained higher levels of intracellular bacilli. Extended necrosis was observed in granulomas from M. tuberculosis- but not bacillus Calmette–Guérin-infected humanized mice. Our data indicate that humanized mice can be used as a model to study the formation and maintenance of human granuloma in tuberculosis and other infectious or noninfectious diseases.

Keywords: BCG, IFN-γ, TNF-α, CD34

Approximately one third of the human population is infected by M. tuberculosis, of whom 5 to 15% develop active tuberculosis (TB) during their lifetime (1). Mouse models have contributed to our understanding of the pathogenesis and immunology of TB. However, the relative hierarchy of immune-mediated killing mechanisms in humans is still unclear. Moreover, the mouse model fails to form granulomas that reproduce those seen in humans (2). Typical mouse lesions lack central necrosis and are less organized than TB lesions found in immune-competent humans. On the contrary, experimental studies of human immunity to mycobacterial infections are limited by obvious practical and ethical restrictions.

The host counters mycobacterial infections primarily via CD4+ T helper1 (Th1) cell-mediated immune responses involving cellular effector mechanisms resulting in macrophage activation. In murine models of mycobacterial infection, CD4+ T cells have been shown to be required for the formation of granulomas (3).

TNF, a major regulator of macrophage activation, apoptosis, chemokine and cytokine production, and cellular recruitment via transendothelial migration, is critical for host responses to infection with M. tuberculosis, including granuloma formation in mice (4–6).

Nonobese diabetic NOD/SCID/γcR−/− (NSG) mice and BALB/c Rag2−/−/γcR−/− (BRG) engrafted with human CD34+ hematopoietic stem cells show a de novo differentiation of myeloid and lymphoid cell populations (7–9). These humanized mice have been shown to be useful tools to examine different microorganisms that involve human hematopoietic cells in their life cycles (10).

Here we studied the outcome of infection with attenuated Mycobacterium bovis bacillus Calmette–Guérin and virulent M. tuberculosis, pathogens that infect myeloid cells, in humanized mice. Humanized mice infected with bacillus Calmette–Guérin showed dysfunctional T-cell responses and lack of bacterial control. Notwithstanding abnormal T-cell responses, mice show organized granulomas containing human T cells and macrophages that resemble lesions observed in human TB. TNF controlled bacterial load within granulomas as well as the severity of inflammation, and human CD4+ but not CD8+ T cells were required for granuloma formation in humanized mice.

Results

Outcome of Infection with M. bovis Bacillus Calmette–Guérin in Humanized Mice.

Confirming previous reports (11, 12), blood from NSG mice 10 wk after inoculation of CD34+ cells contained a higher frequency of human CD45+ cells compared with BRG mice treated or not with the myeloablative compound busulfan to increase engraftment efficiency (Fig. S1A). The frequency of CD3+ within CD45+ cells was also higher in NSG than in BRG mice (Fig. S1B). NSG mice were thus chosen as recipients in our humanized model. Spleen and lymph nodes from NSG mice showed CD4+ and CD8+ T cells and naive mature B cells (Fig. S1 C–F). Splenic T cells from humanized mice proliferated and secrete cytokines when stimulated with a mitogen or in an allogeneic mixed lymphocyte reaction (MLR) as previously shown (8).

Humanized mice were infected i.v. with 106 M. bovis bacillus Calmette–Guérin and killed 4 wk after infection. Whereas the frequency of CD45+ and CD3+ cells in spleens from infected and uninfected mice remained similar (Fig. S2 A and B), the CD4+/CD8+ T-cell ratio was higher (Fig. S2 C and D). The majority of CD4+ and CD8+ T cells differentiated from a naive (CD45RA+CCR7+) into an effector-memory (CD45RA−CCR7−) phenotype after infection (Fig. S2 E–G). The frequency of IFN-γ– and/or TNF-secreting CD4+ and CD8+ spleen T cells in response to a polyclonal stimulation was higher in infected compared with uninfected mice (Fig. S2 H–K).

The percentage of human CD45+ cells in lungs and livers and the frequency of CD3+ within CD45+ human cells sharply increased after bacillus Calmette–Guérin infection (Fig. 1 A, B, E, and F). In contrast to observations in spleens, the CD4+/ CD8+ lung T-cell ratio in infected or uninfected mice was similar (Fig. 1 C, D, G, and H).

Fig. 1.

Fig. 1.

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Defective mycobacterial control in humanized mice. Frequencies of CD45+ cells (A and E), of CD3+ within CD45+ cells (B and F), and of CD4+ (C and G) and CD8+ (D and H) within CD3+ cells in lungs and livers of bacillus Calmette–Guérin-infected and control humanized mice were analyzed by FACS. The mean and SEM for each group are depicted. Differences between groups were significant (*P < 0.05 and **P < 0.01, Student t test). The cfu counts in lungs (I) and livers (J) from humanized or nonhumanized mice were determined 4 w after infection with bacillus Calmette–Guérin. The horizontal bar indicates the median value. Differences between groups are significant (*P < 0.05, **P < 0.01, and ***P < 0.001, Mann–Whitney U test). The percentage of PD-1+ (K, L, O, and P) and CD57+ (M, N, Q, and R) CD4+ or CD8+ cells in lungs (K, M, O, and Q) or spleens (L, N, P, and R) from bacillus Calmette–Guérin-infected or uninfected controls are depicted. The mean and SEM for each group (n = 5) are depicted. Differences between groups are significant (*P < 0.05, Student t test).

Surprisingly, higher bacterial loads were observed in the lung and liver of humanized mice compared with nontransplanted controls (Fig. 1 I and J). We speculated that T-cell dysfunctions could account for the lack of T-cell control of bacterial growth. The engagement of Programmed Death-1 (PD-1) protein on activated T-cells down-regulates T-cell function (13). We found that the percentage and expression levels of PD-1–expressing CD4+ and CD8+ lung or spleen cells increased after bacillus Calmette–Guérin infection (Fig. 1 K, L, O, and P and Fig. S3 A–D). Also, CD57 expression in T lymphocytes has been recognized as a marker of in vitro replicative senescence (14). The percentage of CD57+ within CD4+ (but not in CD8+ T cells) was higher in lungs and spleens from infected humanized mice (Fig. 1 M, N, Q, and R). The levels of expression of CD57 in infected and uninfected T cells were similar (Fig. S3 E–H).

Whether a higher bacterial uptake or intracellular growth in human compared with mouse macrophages associates with the higher susceptibility of humanized mice to bacillus Calmette–Guérin infection was then studied. Bacillus Calmette–Guérin uptake by human macrophages was slightly higher in human than in mouse macrophages (Fig. S4A). Human macrophages also contained higher bacterial levels than mouse macrophages at 4 d after infection. The titers of IFN-γ–regulated human GP91 PHOX and INOS mRNA that code for molecules that mediate mycobacterial control by phagocytes were similar in organs from infected and control mice (Fig. S4 B–E).

Granuloma Formation in Bacillus Calmette–Guérin-Infected Humanized Mice.

The histopathological features of the lungs and livers from bacillus Calmette–Guérin-infected humanized mice were then studied. Livers showed rather typical tuberculoid round-shaped granulomas composed of a core of human CD68+ macrophages, surrounded by CD3+ T cells (Fig. 2 A, E, and F). The organized structure of the granuloma was less obvious in lungs (Fig. 2B). Granulomas were observed in neither uninfected humanized mice nor bacillus Calmette–Guérin-infected nonhumanized NSG mice (Fig. 2 C and D). A rim of fibroblasts and a collagen mantle surrounded the granulomas (Fig. 2 A and G). Multinucleated giant cells characteristic of human granulomas were also present (Fig. 2H). Few to moderate numbers of apoptotic and necrotic cells were also observed in the granulomas of humanized mice. Acid fast staining of bacteria in tissue sections indicated that bacillus Calmette–Guérin preferentially localized as single cells within the granuloma (Fig. 2 I and J and Fig. S4F). Moderate to abundant numbers of mouse CD45+ cells were present mainly in the periphery of the granulomas and did not colocalize with bacillus Calmette–Guérin (Fig. S4 G and H).

Fig. 2.

Fig. 2.

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Granuloma formation in bacillus Calmette–Guérin-infected humanized mice. Inflammatory lesions in liver (A) and lung (B) from humanized mice 4 wk after infection with bacillus Calmette–Guérin observed in sections stained with H&E. The number of inflammatory foci in livers and lungs (C) and the percentage of total lung area occupied with lesions in infected humanized mice are compared with uninfected humanized mice or infected nonhumanized controls (D). The mean ± SEM in at least five mice per group are depicted. The distribution of CD3+ T cells (E) and CD68+macrophages (F) in liver granulomas was determined by immunohistochemistry. A collagenic mantle surrounding the granuloma was visualized by using Sirius red staining (G). (H) Micrograph of a giant cell within a granuloma in an H&E-stained section. Localization of acid-fast bacilli identified by Ziehl–Nielsen staining (arrows) within the granuloma in the liver of humanized mice at magnifications of 400× (I) and 1,000× (J).

Transcript levels of human IFN-γ as well as of IFN-γ–regulated T-cell chemotactic molecules CXCL9 and CXCL10 were increased in the lungs and livers of bacillus Calmette–Guérin-infected humanized mice compared with those from noninfected animals (Fig. 3 A–F). A trend to increased titers of monocyte chemotactic CCL2 mRNA was observed in livers from infected mice (Fig. 3G).

Fig. 3.

Fig. 3.

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Human CD4+ cells are required for granuloma formation in bacillus Calmette–Guérin-infected humanized mice. The mean levels of IFN-γ (A and D), CXCL9 (B and E), CXCL10 (C and F), and CCL2 (G) mRNA normalized to human β-2 microglobulin housekeeping gene (β2M) transcripts were measured by real-time PCR in lungs (A–C) and livers (D–G) from bacillus Calmette–Guérin-infected or uninfected humanized mice. Differences between groups are significant (*P < 0.05, **P < 0.01, and ***P < 0.001, Student t test). Humanized mice were inoculated i.p. with 100 μg anti-human CD4 or 50 μg anti-CD8 mAbs for three consecutive days starting 3 d before and 14 d after bacillus Calmette–Guérin inoculation. The mean histopathological scoring of H&E-stained liver sections (H) and the relative score of CD3+ or CD68+ cells in liver granulomas from mAb-treated or control humanized mice are depicted (I). Differences between groups are significant (*P < 0.05, **P < 0.01 and ***P < 0.001 Student t test). Micrographs of H&E-stained liver sections from control (J) or CD4+ cell-depleted (K) humanized mice 4 wk after inoculation. Arrows indicate granulomas. The cfu counts in lungs (L) and livers (M) from humanized mice treated with anti-CD4 or anti-CD8 mAbs or nonhumanized NSG mice were determined 4 wk after bacillus Calmette–Guérin infection. Quartile boxes and 10th to 90th percentile whiskers are depicted. Differences between groups are significant (*P < 0.05, **P < 0.01, and ***P < 0.001, Mann–Whitney U test).

Role of CD4+ Cells and TNF in Granuloma Formation in Bacillus Calmette–Guérin-Infected Humanized Mice.

Whether CD4+ and CD8+ cells participated in granuloma formation was then studied. The administration of anti-CD4 and anti-CD8 mAbs before and during bacillus Calmette–Guérin infection reduced the respective T-cell population (Fig. S5 A–F). Livers from CD4+ cell-depleted mice showed fewer, minimal, and irregular foci, consisting of macrophages and some neutrophils, whereas CD3+ cells were occasional or absent (Fig. 3 H–K). On the contrary, CD8+ cell-deficient humanized mice retained the granuloma numbers and their complex structure and size (Fig. 3H). Few human CD8+ cells were found within the granulomas of nondepleted humanized mice (Fig. S5G).

Bacillus Calmette–Guérin levels in lungs or livers of anti-CD4–treated (but not anti-CD8–treated) humanized mice were reduced compared with nontreated humanized mice (Fig. 3 L and M).

TNF is required for granuloma formation in mice infected with mycobacteria (4). Humanized mice showed increased levels of human and mouse TNF-α mRNA after infection with bacillus Calmette–Guérin (Fig. 4 A and B). Therefore, we studied the effect of administration of the TNF inhibitor etanercept on granuloma formation in bacillus Calmette–Guérin-infected humanized mice. Etanercept, a recombinant TNF p75 receptor containing protein that inhibits the signaling via soluble or membrane bound TNF (15), is used for treatment of different autoimmune diseases (16). Spleens from etanercept-treated infected mice showed a slightly increased CD4+/ CD8+ cell ratio, but similar proportions of CD45+ and CD3+ cells as controls (Fig. S6 A–D). The frequencies of CD45RA-, CCR7-, CD57-, or PD-1–expressing CD4+ or CD8+ cells in spleens from etanercept-treated and untreated infected mice were similar (Fig. S6 E–J).

Fig. 4.

Fig. 4.

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Effect of treatment with the TNF inhibitor etanercept in the outcome of infection of humanized mice with bacillus Calmette–Guérin. The mean levels of human (A) and mouse (B) TNF-α mRNA normalized to human β2M transcripts were measured by real-time PCR in liver from bacillus Calmette–Guérin-infected or uninfected humanized mice. Differences between groups are significant (**P < 0.01, Student t test). Mice were treated i.p. with 0.125 mg/kg etanercept every other day starting 1 d before bacillus Calmette–Guérin infection during 4 wk. The mean severity of inflammation score in H&E-stained liver (C) and lung (D) sections from etanercept-treated or control humanized mice are depicted (n > 6 mice per group). The mean acid-fast bacilli density score in Ziehl–Nielsen-stained liver (E) and lung (F) sections from etanercept-treated or control humanized mice are depicted (n > 6 mice per group). Representative micrographs showing the acid fast bacteria (AFB) density and their localization within the granuloma of from liver sections treated with etanercept or not treated are shown (G and H). The cfu counts in livers (I and K) and lungs (J and L) from humanized (I and J) or nonhumanized NSG (K and L) mice treated with etanercept were determined 4 wk after bacillus Calmette–Guérin infection. Quartile boxes and 10th to 90th percentile whiskers are depicted. Differences between groups are significant (*P < 0.05, **P < 0.01, and ***P < 0.001, Mann–Whitney U test).

Livers (but not lungs) from etanercept-treated humanized mice displayed milder inflammatory responses (Fig. 4 C and D), whereas the histological structure of the granuloma was preserved.

Bacterial aggregates were observed in granulomas from etanercept-treated mice, whereas granulomas from control mice showed lower numbers of bacteria usually as single cells or smaller aggregates (Fig. 4 E–H). Accordingly, livers and lungs from etanercept-treated humanized mice contained higher cfu levels than controls (Fig. 4 I and J), whereas bacterial levels in etanercept-treated and control nonhumanized NSG mice were similar (Fig. 4 K and L). Levels of TNF-α mRNA in livers from control and anti-CD4–treated or anti-CD8–treated infected mice were similar (Fig. S6K).

Granuloma Formation in M. tuberculosis-Infected Humanized Mice.

The outcome of aerosol infection with 250 M. tuberculosis bacteria in humanized mice was studied next. Similar to observations in mice infected with bacillus Calmette–Guérin, IFN-γ, CXCL9, and CXCL10 mRNA levels in lungs and livers from humanized mice were elevated after M. tuberculosis infection (Fig. 5 A–F). Bacterial levels in humanized or control NSG mice were strikingly increased compared with those in bacillus Calmette–Guérin-infected mice (more than 3 and 1 log in lungs and livers respectively). Higher bacterial titers were quantified in the liver from humanized mice compared with nonhumanized controls (Fig. 5G).

Fig. 5.

Fig. 5.

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Granuloma formation in M. tuberculosis-infected humanized mice. Total RNA was extracted from lungs (A–C) and livers (D–F) from humanized mice 4 wk after M. tuberculosis infection or from uninfected controls. The IFN-γ (A and D), CXCL9 (B and E), and CXCL10 (C and F) mRNA levels normalized to human β2M were assessed by real-time PCR. The horizontal lines are the mean for each group. Differences between groups are significant (*P < 0.05, **P < 0.01, and ***P < 0.001, Student t test). Quartile boxes and 10th to 90th percentile whiskers depict cfu counts in organs from humanized mice 4 wk after infection with M. tuberculosis (G). Differences between groups (n ≥ 8) are significant (*P < 0.05, Mann–Whitney U test). Histopathological scoring of H&E-stained liver (H) and lung (I) sections from humanized and control mice infected with M. tuberculosis. The score of severity of lesions, the presence of necrosis, and macrophage cellularity in the granulomas in lung and liver are depicted. Differences between groups are significant (*P < 0.05 and **P < 0.01, Student t test). H&E staining shows representative inflammatory lesions in the liver from humanized (J) and control NSG (K) mice 4 wk after infection with M. tuberculosis. (Magnification: 400×.) (L) Micrograph of CD3+ stained cells in the periphery of a liver granuloma from M. tuberculosis-infected humanized mouse.

Nonhumanized mice infected with M. tuberculosis (but not with bacillus Calmette–Guérin) showed granulomatous lesions in livers (Fig. 5K and Fig. S7A). However, the pathology was significantly more severe in humanized mice (Fig. 5 H and J and Fig. S7 B and C). Small and irregular microscopic granulomatous lesions formed by macrophages and epithelioid cells were observed in livers from NSG control mice. Instead, humanized mice showed small and large macroscopic lesions, the latter irregular or round in shape. The large lesions showed CD3+ cells in the periphery (Fig. 5L) and were occasionally surrounded by a collagen layer (Fig. S7D). Giant cells were observed in the center of the granulomas. An extensive necrosis was present in liver lesions from humanized but not in nonhumanized mice (Fig. 5 H, J, and K and Fig. S7 A and B).

Humanized and control mice showed similar severity of lesions in the lungs. However, lung granulomas from humanized mice showed increased necrotic areas compared with nonhumanized controls (Fig. 5I and Fig. S7 E and F).

Discussion

Here we demonstrate that, despite impaired bacterial control, humanized mice showed organized granulomas when infected with bacillus Calmette–Guérin or M. tuberculosis. Human CD4+ but not CD8+ cells were required for granuloma formation in mycobacteria-infected humanized mice. The control of mycobacteria in granulomas and the severity of inflammatory responses were reduced by inoculation of a TNF inhibitor, but the granuloma structure was preserved.

The majority of CD4+ and CD8+ T cells from mycobacterial-infected humanized mice differentiated into an effector-memory phenotype, resembling the generalized immune activation observed in homeostatic peripheral T-cell expansion during lymphopenic condition, which is accompanied by a decreased threshold for T-cell receptor activation (17). Surprisingly, higher bacterial levels were detected in humanized mice compared with controls, which could be reverted by depletion of human CD4+, but not CD8+, cells.

The majority of T cells from bacillus Calmette–Guérin-infected humanized mice also expressed high levels of PD-1. PD-1 limits proliferation, increases apoptosis, and interferes with effector functions of T cells against infection with M. tuberculosis in man (18). Increased expression of CD57, a marker of T-cell clonal exhaustion, was also detected in CD4+ cells of infected humanized mice. The augmented expression of PD-1 and CD57 probably reflect dysfunctional T-cell responses in infected humanized mice that might underlie their defective mycobacterial control.

The granuloma, which is the classic pathological feature of TB, is the niche in which the bacillus can grow or persist, and the microenvironment in which immune cells interact to prevent mycobacterial dissemination (19). The strength of our humanized model lies in the formation of granulomas that resemble those observed in human mycobacteriosis. Granulomas showed a core with large numbers of human CD68+ macrophages, giant multinucleated cells, and higher density of bacilli compared with that in surrounding tissues. A layer of lymphocytes and fibroblasts surrounded the core. Particularly in the liver, these lesions were more organized and sphere-like than those formed during mouse M. tuberculosis infection (20). The accumulation of fibroblasts and a collagen capsule occurs in human benign evolution of M. tuberculosis infection (21).

Consistent with granuloma formation, organs from infected humanized mice showed a dramatic accumulation of human CD45+ cells and an augmented expression of IFN-γ, CXCL9, and CXCL10, as well as CCL2, chemokines shown to participate in the formation of granulomas in mouse models (22, 23).

CD4+ but not CD8+ cells were required for granuloma formation in bacillus Calmette–Guérin-infected humanized mice. Similarly, in MhcII−/− or Cd4−/− mice, CD4+ T cells have been shown to contribute to the organization of granulomas in M. tuberculosis-infected mice (24, 25).

Whether CD4+ T cells regulate granuloma formation in humanized mice results from the cognate interactions of antigen-specific cells is uncertain. Low-level T-cell responses against viral infections were induced in some cases (8, 26), but are absent in other reports in humanized mice (27, 28). In our model, T cells are positively selected by mouse MHC and might not function well in a HLA-restricted manner (29). Non–mycobacterial-specific T cells might account for granuloma formation in humanized mice because (i) the adoptive transfer of monoclonal, nonbacterial specific CD4+ T cells restores the granulomatous response to bacillus Calmette–Guérin infection of Rag1−/− mice, but not the bacterial control (30); and (ii) mycobacteria-specific T cells are hardly more arrested than T cells of other specificities in the granuloma (31).

The treatment of humanized but not control mice with the TNF inhibitor etanercept resulted in a larger number of bacilli and reduced severity of inflammation, confirming studies on the role of TNF in mycobacterial containment in conventional mouse models (5, 32). However, in contrast to murine models (32), the granuloma structure was preserved in etanercept-treated humanized mice. TNF neutralization in nonhuman primates resulted in disseminated TB but a normal granuloma structure (33). Thus, although the human transplant is required for granuloma formation during bacillus Calmette–Guérin infection, TNF controls bacterial levels in the granuloma of humanized mice.

Of importance, human and mouse TNF and TNF receptors are functionally cross-reactive (34), and etanercept also inhibits mouse TNF (35). Human and murine TNF-α mRNA levels were increased in organs from humanized bacillus Calmette–Guérin-infected mice. However, the titers of bacillus Calmette–Guérin in the liver or lung from NSG mice inoculated with etanercept were similar, suggesting that etanercept inhibits human TNF in humanized mice. Accordingly, M. tuberculosis-infected mice treated with higher concentrations of etanercept than described here showed similar bacterial load as controls (36).

Despite the expression of IFN-γ, levels of human INOS and GP91PHOX mRNA did not increase after infection. Thus, the expression of iNOS or gp91phox in humanized mice has different requirements than that of CXCL9 or CXCL10, which were up-regulated after infection. In relation, treatment of patients with TB with aerosolized IFN-γ increased CXCL10 but not iNOS levels in lung cells (37). The failure to induce these microbicidal mechanisms, and the superior bacterial uptake by human compared with mouse macrophages, might also underlie the lack of protection against bacillus Calmette–Guérin infection in humanized mice.

Nonhumanized mice infected with M. tuberculosis showed granulomatous lesions, whereas these were not observed in bacillus Calmette–Guérin-infected mice. An accelerated granuloma formation in response to M. tuberculosis compared with bacillus Calmette–Guérin has been described (38). RD1, a virulence region that is absent in bacillus Calmette–Guérin, has been shown to participate in granuloma formation (39, 40). Aggregates of epithelioid macrophages have been previously observed in mycobacterial-infected SCID mice (41, 42). However, humanized mice showed granulomas with a more organized structure and an increased severity of the size and area occupied by the lesions.

Large necrotic areas were observed in lesions from M. tuberculosis-infected humanized mice but not in bacillus Calmette–Guérin-infected humanized mice or M. tuberculosis-infected nonhumanized mice. The necrosis was coagulative rather than liquefactive, and the dead tissue architecture was preserved. Supporting our data, M. tuberculosis stimulates necrosis, allowing viable bacilli to escape from host cells, after which they can infect new cells and elicit inflammation (43–45). T cells are required for necrosis in granulomas of Mycobacterium avium-infected mice (46). However, whether human T cells mediate necrosis in granulomas from M. tuberculosis-infected humanized mice remains to be studied.

Granuloma initiation has been interpreted as a host-protective event, to provide the microenvironment in which specific T cells activate macrophages to contain M. tuberculosis infection (20). On the contrary, our results suggest that dysfunctional CD4+ T cells participate in granuloma formation in humanized mice and may facilitate bacterial growth. Whether such a conclusion can be generalized for the natural course of infection requires further studies. Data in a zebrafish model suggest that mycobacteria exploit the granuloma for local expansion and systemic dissemination (47).

Thus, the humanized mouse model can be used to study the role of cytokines, chemokines, different immune cells, the effect of HIV coinfection, host genetics, and bacterial components in mycobacterial granuloma formation. We also propose the use of humanized mice as a model for other infectious and noninfectious granulomatous diseases.

Materials and Methods

All animal experiments were conducted in accordance with guidelines of Karolinska Institute, and approved by Stockholm's District Ethical Committee of Animal Research. The procedures for generation and infection of humanized mice with mycobacteria are described in Supporting Information. The quantification of transcripts in organs by real-time PCR, the staining of surface receptors and intracellular cytokines, the histopathological analysis, and the generation of human and mouse macrophages are explained in SI Materials and Methods.

Supplementary Material

Supporting Information
supp_110_16_6482__index.html (7KB, html)

Acknowledgments

We thank Dr. Francesca Chiodi, Dr. Antonio Rothfuchs, and Dr. Camille Locht for comments on the manuscript; Ms. Margareta E. Andersson and Mr. Kenth Andersson for excellent technical assistance; and Ms. Ewa Westergren for the preparation of histological slides. This work was supported by a European Community 200732 HOMITB (Host Mycobacterial Interactions in Immunity and Pathogenesis of Tuberculosis) Grant, European Community Marie Curie HIV-TB (HIV and Tuberculosis infections) project, the Karolinska Institutet, and the Swedish Research Council.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1219985110/-/DCSupplemental.

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