Dendritic Cells in Chronic Mycobacterial Granulomas Restrict Local Anti-Bacterial T Cell Response in a Murine Model

Background

Mycobacterium-induced granulomas are the interface between bacteria and host immune response. During acute infection dendritic cells (DCs) are critical for mycobacterial dissemination and activation of protective T cells. However, their role during chronic infection in the granuloma is poorly understood.

Methodology/Principal Findings

We report that an inflammatory subset of murine DCs are present in granulomas induced by Mycobacteria bovis strain Bacillus Calmette-guerin (BCG), and both their location in granulomas and costimulatory molecule expression changes throughout infection. By flow cytometric analysis, we found that CD11c⁺ cells in chronic granulomas had lower expression of MHCII and co-stimulatory molecules CD40, CD80 and CD86, and higher expression of inhibitory molecules PD-L1 and PD-L2 compared to CD11c⁺ cells from acute granulomas. As a consequence of their phenotype, CD11c⁺ cells from chronic lesions were unable to support the reactivation of newly-recruited, antigen 85B-specific CD4⁺IFNγ⁺ T cells or induce an IFNγ response from naïve T cells in vivo and ex vivo. The mechanism of this inhibition involves the PD-1:PD-L signaling pathway, as ex vivo blockade of PD-L1 and PD-L2 restored the ability of isolated CD11c⁺ cells from chronic lesions to stimulate a protective IFNγ T cell response.

Conclusions/Significance

Our data suggest that DCs in chronic lesions may facilitate latent infection by down-regulating protective T cell responses, ultimately acting as a shield that promotes mycobacterium survival. This DC shield may explain why mycobacteria are adapted for long-term survival in granulomatous lesions.     

Variability in Tuberculosis Granuloma T Cell Responses Exists,but a Balance of Pro- and Anti-inflammatory Cytokines Is Associated with Sterilization

Lung granulomas are the pathologic hallmark of tuberculosis (TB). T cells are a major cellular component of TB lung granulomas and are known to play an important role in containment of Mycobacterium tuberculosis (Mtb) infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB with clinically active disease, latent infection or early infection, to understand functional characteristics and dynamics of T cells in individual granulomas. We sought to correlate T cell cytokine response and bacterial burden of each granuloma, as well as granuloma and systemic responses in individual animals. Our results support that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst animals with different clinical status, indicating that a diversity of granulomas exists within an individual host. On average only about 8% of T cells from granulomas respond with cytokine production after stimulation with Mtb specific antigens, and few “multi-functional” T cells were observed. However, granulomas were found to be “multi-functional” with respect to the combinations of functional T cells that were identified among lesions from individual animals. Although the responses generally overlapped, sterile granulomas had modestly higher frequencies of T cells making IL-17, TNF and any of T-1 (IFN-γ, IL-2, or TNF) and/or T-17 (IL-17) cytokines than non-sterile granulomas. An inverse correlation was observed between bacterial burden with TNF and T-1/T-17 responses in individual granulomas, and a combinatorial analysis of pair-wise cytokine responses indicated that granulomas with T cells producing both pro- and anti-inflammatory cytokines (e.g. IL-10 and IL-17) were associated with clearance of Mtb. Preliminary evaluation suggests that systemic responses in the blood do not accurately reflect local T cell responses within granulomas.     

Helical Carbon Nanotubes Enhance the Early Immune Response and Inhibit Macrophage-Mediated Phagocytosis of Pseudomonas aeruginosa

Aerosolized or aspirated manufactured carbon nanotubes have been shown to be cytotoxic, cause pulmonary lesions, and demonstrate immunomodulatory properties. CD-1 mice were used to assess pulmonary toxicity of helical carbon nanotubes (HCNTs) and alterations of the immune response to subsequent infection by Pseudomonas aeruginosa in mice. HCNTs provoked a mild inflammatory response following either a single exposure or 2X/week for three weeks (multiple exposures) but were not significantly toxic. Administering HCNTs 2X/week for three weeks resulted in pulmonary lesions including granulomas and goblet cell hyperplasia. Mice exposed to HCNTs and subsequently infected by P. aeruginosa demonstrated an enhanced inflammatory response to P. aeruginosa and phagocytosis by alveolar macrophages was inhibited. However, clearance of P. aeruginosa was not affected. HCNT exposed mice depleted of neutrophils were more effective in clearing P. aeruginosa compared to neutrophil-depleted control mice, accompanied by an influx of macrophages. Depletion of systemic macrophages resulted in slightly inhibited bacterial clearance by HCNT treated mice. Our data indicate that pulmonary exposure to HCNTs results in lesions similar to those caused by other nanotubes and pre-exposure to HCNTs inhibit alveolar macrophage phagocytosis of P. aeruginosa. However, clearance was not affected as exposure to HCNTs primed the immune system for an enhanced inflammatory response to pulmonary infection consisting of an influx of neutrophils and macrophages.     

Neutrophils in Oral Paracoccidioidomycosis and the Involvement of Nrf2

Neutrophils have been implicated in granuloma formation in several infectious diseases, in addition to their main phagocytic and pathogen destruction role. It has been demonstrated that Nrf2 regulates antioxidant protection in neutrophils, attenuating inflammation without compromising the hosts bacterial defense. In this study, we analyzed the presence of neutrophils in Paracoccidioides brasiliensis mycosis (PCM), as well as the immunoexpression of Nrf2. Thirty-nine cases of oral PCM were classified according to quantity of fungi and to the presence of loose or well-organized granulomas and microabscesses. An Nrf2 antibody was used for immunohistochemical analysis. The results showed that neutrophils are present in microabscesses and loose granulomas, but were absent in structured granulomas. A greater quantity of fungi was shown in cases with only loose granulomas when compared to loose and well organized granulomas. Nrf2 was observed in the nuclei of neutrophils of loose granulomas and abscesses, with its expression in loose granulomas maintained despite the additional presence of well organized granulomas in the same specimen. This study suggests that neutrophils participate in P. brasiliensis granuloma formation and that Nrf2 has a possible role in neutrophil survival, via modulation of the inflammatory response.     

Pasteurellosis as a cause of genital lesions in rams. A descriptive study

Pasteurellosis is one of the most prevalent diseases of sheep, but the involvement of Pasteurellae in genital pathology of rams has been described rarely. One hundred and eighty-four rams showing palpable lesions in testes, epididymides or scrotum were submitted to bacteriological studies, and seven mature rams found infected with bacterial species belonging to the Pasteurella cluster (i.e., Mannheimia, Pasteurella and Bibersteinia (M/P/B)). The M/P/B cultures obtained were pure and/or heavy, and were confirmed after necropsy in the five M/P/B infected rams that could be slaughtered for further pathological examinations. Pasteurella multocida infected rams exhibited fibrinous exudate and generalized adhesions between the vaginal and the external scrotal layers. Testicular atrophy and epididymal sperm granulomas were also evident in these rams. Microscopically, epithelial hyperplasia with intraepithelial cysts, fibrosis and spermatic granulomas were present in the epididymis, while testis showed sperm stasis foci, microcalcifications and fibrosis. Mannheimia haemolytica infected rams showed severe unilateral epididymitis and testicular atrophy, being microscopically similar to the lesions found in P. multocida infected rams. The ram found infected with B. threalosi had severe unilateral lesions in testis, epididymis and scrotum. Microscopically, abscesses in epididymis and testis, and severe fibrosis and interstitial round cells infiltrates in testis were observed. Further studies should be conducted to determine properly the role played by the Pasteurella cluster in the pathogenesis of genital lesions in rams.     

Epstein-Barr Virus Infection in Chronically Inflamed Periapical Granulomas

Periapical granulomas are lesions around the apex of a tooth caused by a polymicrobial infection. Treatment with antibacterial agents is normally performed to eliminate bacteria from root canals; however, loss of the supporting alveolar bone is typically observed, and tooth extraction is often selected if root canal treatment does not work well. Therefore, bacteria and other microorganisms could be involved in this disease. To understand the pathogenesis of periapical granulomas more precisely, we focused on the association with Epstein-Barr virus (EBV) using surgically removed periapical granulomas (n = 32). EBV DNA was detected in 25 of 32 periapical granulomas (78.1%) by real-time PCR, and the median number of EBV DNA copies was approximately 8,688.01/μg total DNA. In contrast, EBV DNA was not detected in healthy gingival tissues (n = 10); the difference was statistically significant according to the Mann-Whitney U test (p = 0.0001). Paraffin sections were also analyzed by in situ hybridization to detect EBV-encoded small RNA (EBER)-expressing cells. EBER was detected in the cytoplasm and nuclei of B cells and plasma cells in six of nine periapical granulomas, but not in healthy gingival tissues. In addition, immunohistochemical analysis for latent membrane protein 1 (LMP-1) of EBV using serial tissue sections showed that LMP-1-expressing cells were localized to the same areas as EBER-expressing cells. These data suggest that B cells and plasma cells in inflamed granulomas are a major source of EBV infection, and that EBV could play a pivotal role in controlling immune cell responses in periapical granulomas.     

Capillaria hepatica: granuloma formation to eggs. II. Peripheral immunological responses

Peripheral immunological responses were assessed by indirect hemagglutination, passive cutaneous anaphylaxis, gel diffusion, and delayed dermal reactivity in mice with experimental primary and secondary Capillaria hepatica egg granulomas. Agglutinating and homocytotropic antibodies as well as delayed dermal reactivity, but not precipitating antibodies, were detected in animals with primary and secondary granulomas. The demonstration of circulating antibody during the course of granuloma formation indicates a possible role for antibody in the response and is cause for a reassessment of the etiology of experimental helminth egg granulomas.     

Development of spermatic granuloma in albino rats following administration of water extract of Heliotropium bacciferum Forssk

A spermatic granuloma is a chronic inflammatory reaction produced in response to extravasated sperm within the intertubular connective tissue. The present study investigates the possible toxic effects of water extract of Heliotropium bacciferum on the reproductive system of male albino rats and the associated potential for the development of spermatic granulomas. H. bacciferum is a herbal plant used in traditional medicine and reported to have cytotoxic effects due to pyrrolizidine alkaloids. Histological examinations revealed no changes in the tissues of the testes, although, some changes were detected in the cauda epididymis, the most important of which was the development of small lesions of spermatic granulomas. Clear gaps were observed between the epithelial linings of the epididymal tubules.     

A model of T-cell unresponsiveness using the male-specific antigen, H-Y

Granuloma formation in schistosomiasis japonica differs in several respects from those observed in Schistosoma mansoni infections. We have utilized the lung granuloma model in mice sensitized with subcutaneous injection of Schistosoma japonicum eggs to study the kinetics and mechanisms of this response. Animals injected subcutaneously with a range of 50–50,000 S. japonicum eggs elicited a significant pulmonary granulomatous response around ova subsequently injected intravenously. The pulmonary granulomas were formed of macrophages, lymphocytes, and eosinophils. Both antithymocyte globulin and antieosinophil sera reduced significantly the size of the granulomas and depleted the corresponding cell. Nude athymic mice developed markedly reduced pulmonary granulomas as did mice treated with niridazole or hydrocortisone. Sensitization to the egg antigens was demonstrable as both immediate and arthus-type footpad responses. Our data show that cell-mediated pulmonary granulomas can form around S. japonicum eggs in animals previously sensitized by the subcutaneous route. This model may provide further insights into the pathogenesis of S. japonicum granuloma.     

Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant

Granulomas are organized host immune structures composed of tightly interposed macrophages and other cells that form in response to a variety of persistent stimuli, both infectious and noninfectious. The tuberculous granuloma is essential for host containment of mycobacterial infection, although it does not always eradicate it. Therefore, it is considered a host-beneficial, if incompletely efficacious, immune response. The Mycobacterium RD1 locus encodes a specialized secretion system that promotes mycobacterial virulence by an unknown mechanism. Using transparent zebrafish embryos to monitor the infection process in real time, we found that RD1-deficient bacteria fail to elicit efficient granuloma formation despite their ability to grow inside of infected macrophages. We showed that macrophages infected with virulent mycobacteria produce an RD1-dependent signal that directs macrophages to aggregate into granulomas. This Mycobacterium-induced macrophage aggregation in turn is tightly linked to intercellular bacterial dissemination and increased bacterial numbers. Thus, mycobacteria co-opt host granulomas for their virulence.     

Brugia malayi,Brugia pahangi, and Brugia patei: Pulmonary pathology in jirds, Meriones unguiculatus

The chronological development of pulmonary lesions due to subperiodic Brugia malayi and related species was studied in the jird, Meriones unguiculatus. Major pathologic changes included (1) granulomas induced by larvae and adults, (2) obstructive endarteritis, and (3) chronic interstitial inflammation with degenerating microfilariae. The majority of pulmonary granulomas were initiated near the time of the final molt, about 30 days postinoculation, followed by involution and formation of residual vascular lesions over the next several months. A minority of granulomas arose about sexually mature adult worms and showed a characteristic sequence of development along the length of these worms. Ultrastructural observations suggested that within granulomas internal structures of the worm underwent an autolytic-like disintegration, while the cuticle remained intact. A material, presumably of parasitic origin, then appeared between the cuticle and adherent epitheloid and giant cells and was subsequently phagocytized by these cells. Obstructive endarteritis appeared to peak at about the time of the final molt, and became largely fibrotic by 125 days postinoculation; electron microscopy of the subintimal infiltrates revealed a variety of cells including inflammatory cells and others interpreted as modfied cells of vascular origin (smooth muscle cells and two types of endothelial cells).Parasitological data suggested that both larvae and adults migrated to the lungs and that mating occurred here, rather than in peripheral sites of development. In terms of development, reproduction and survival, the pulmonary arteries of the male jird offered a suitable alternative for the localization of these primarily lymphatic filariae; our results thus suggest that the pulmonary localization of these worms should not be considered indicative of an aberrant mode of development. The failure of female jirds to develop detectable peripheral microfilaremia was associated with a high rate of infertility among female worms, rather than pulmonary sequestration or destruction of microfilariae.     

Schistosomiasis mansoni, haematobia, and japonica in hamsters: liver granuloma measurements

Differences in the granulomatous response of hamsters infected with Schistosoma mansoni, Schistosoma haematobium, or Schistosoma japonicum were studied by measuring granulomas formed around eggs in the livers at 1, 3, 5, 11, and 19 week intervals after patency of the infections. For each species, the mean diameters of both granulomas containing only 1 egg and granulomas selected at random were determined. The mean volume of single egg granulomas in S. mansoni-infected hamsters was greater than in those with other species at all time intervals studied. The decrease in single egg granuloma size with time occurred more gradually in S. mansoni infections. The mean volume of granulomas measured at random was greatest in S. japoracum-infected animals at 1, 3, and 11 weeks after patency; was greatest in S. haematobium-iufected. hamsters at 5 and 19 weeks; and was least in S. mansoni-infected hamsters at all time periods. During the 19 week period following patency, the mean volume of randomly measured granulomas increased with time in S. haematobium infections, decreased gradually in S. mansoni infections, and decreased markedly in S. japonicum infections. Multi-egg granulomas represented 2–6% of all granulomas measured in S. mansoni infections, 38–68% in S. haematobium infections, and 20–53% in S. japonicum infections. An estimated proportion of the liver occupied by granulomas, i.e., a lesion-to-tissue ratio, was computed. There was no consistent difference in this ratio among the 3 species when the data were grouped in comparable intervals of mean number of eggs per g of liver. The lesion-to-tissue ratio increased with increasing numbers of eggs per g of liver. The host response of hamsters to these 3 species of schistosomes differed markedly even at comparable levels of eggs per g of liver. This study provides further evidence of an intrinsic qualitative difference in the eggs of the 3 species. The species of eggs present in tissues apparently has greater influence on the host response than does the quantity of eggs present.     

Leishmania mexicana: Immunology and histopathology in C3H mice

C3H mice were infected subcutaneously with 10⁵ promastigotes of Leishmania mexicana and subsequent lesions were examined at 3, 5, and 8 months. All animals developed persistent nonulcerating nodules of variable size which did not metastasize. The nodules contained amastigotes with a mononuclear infiltrate of histiocytes, lymphocytes, and plasma cells, but without formation of tuberculoid-type granulomas. Neutrophils and eosinophils were also encountered in some cases. Specific antileishmanial antibodies and delayed-type hypersensitivity to leishmanial antigen were present at 3, 5, and 8 months postinfection. L. mexicana infection in C3H mice differs from classic self-healing cutaneous leishmaniasis by the pesistence of nonhealing, nonulcerating, nonmetastasizing lesions, despite evidence of cellular and humoral immunity.     

Pasteurella Septica Infections in Humans

Five instances of infection by Pasteurella septica, an organism which closely resembles H. influenzae on methods of presumptive bacteriology, are reported from an acute treatment general hospital practice over a period of three months. This infection decidedly outnumbers those caused by other Pasteurella species in North America. The organism is apt to be identified as H. influenzae, Friedlander''s bacillus or Mima polymorpha. Unlike these, it shows extreme sensitivity to penicillin, sometimes combined with resistance to antibiotics that inhibit or kill these other organisms.While in the past Pasteurella septica infection was considered to be characterized by persistent local infection following domestic animal scratches or bites, it is clear that it is more often found in cases of respiratory tract infection and peripheral septic disease. In three of the five cases reported, exposure to animals was the source of infection.Because mixed infection with confusingly similar organisms may occur, lack of awareness of extreme penicillin sensitivity of Pasteurella septica may account for persistent failure of antibiotic therapy.     

Caseating Granulomas in Cutaneous Leishmaniasis

Background

Caseating granulomas are often associated with a mycobacterial infection (TB) and are thought to be exceedingly rare in cutaneous leishmaniasis (CL). However, no large series has accurately documented the incidence of caseating granulomas in CL.

Methods

A multiregional cohort consisting of 317 patients with CL [Syria (157), Pakistan (66), Lebanon (47), Saudi Arabia (43), Ethiopia (2) and Iran (2)] was reviewed. Clinical [age, sex, disease duration, lesion type and geographic and anatomic location] and microscopic data [presence of and type of granuloma, Ridley''s parasitic index (PI) and pattern (RP)] were documented. Presence of microorganisms was evaluated using special stains (GMS, PAS, AFB and Gram) and polymerase chain reaction (PCR) for TB and CL. All cases included in this study were confirmed as CL by PCR followed by restriction fragment length polymorphism analysis for molecular speciation and were negative for other organisms by all other studies performed. Categorical and continuous factors were compared for granuloma types using Chi-square, t-test or Mann-Whitney test as appropriate.

Results

Granulomas were identified in 195 (61.5%) cases of CL and these were divided to 49 caseating (25.2%), 9 suppurative (4.6%) and 137 tuberculoid without necrosis (70.2%). Caseating and tuberculoid granuloma groups were significantly different in terms of the geographical source, with more cases harboring caseating granulomas in Saudi Arabia (p<0.0001). Histologically, both groups were also different in the distribution of their RP (p<0.0001) with a doubling RP3 in caseating granulomas (31% vs. 15%) as opposed to doubling of RP5 in tuberculoid granuloma group (38% vs. 19%). Time needed to achieve healing (RP5) was notably shorter in tuberculoid vs. caseating group (4.0 vs. 6.2 months). Parasitic Index, CL species and other considered variables did not differ for the granuloma type groups.

Conclusion

In our multiregional large cohort, a notable 18.2% of all CL cases harbored caseating granulomas therefore; CL should be considered part of the differential diagnosis for cases with caseating granulomas in endemic regions, especially considering that the regions included in our cohort are also endemic for TB. Of note, cases of CL with caseating granulomas also showed a slower healing process, with no association with specific species, which may be due to worse host immune response in such cases or to a more aggressive leishmania strains.     

Tumor Necrosis Factor and Schistosoma mansoni egg antigen omega-1 shape distinct aspects of the early egg-induced granulomatous response

Infections by schistosomes result in granulomatous lesions around parasite eggs entrapped within the host tissues. The host and parasite determinants of the Schistosoma mansoni egg-induced granulomatous response are areas of active investigation. Some studies in mice implicate Tumor Necrosis Factor (TNF) produced in response to the infection whereas others fail to find a role for it. In addition, in the mouse model, the S. mansoni secreted egg antigen omega-1 is found to induce granulomas but the underlying mechanism remains unknown. We have recently developed the zebrafish larva as a model to study macrophage recruitment and granuloma formation in response to Schistosoma mansoni eggs. Here we use this model to investigate the mechanisms by which TNF and omega-1 shape the early granulomatous response. We find that TNF, specifically signaling through TNF receptor 1, is not required for macrophage recruitment to the egg and granuloma initiation but does mediate granuloma enlargement. In contrast, omega-1 mediates initial macrophage recruitment, with this chemotactic activity being dependent on its RNase activity. Our findings further the understanding of the role of these host- and parasite-derived factors and show that they impact distinct facets of the granulomatous response to the schistosome egg.     

Lack of cathepsin activities alter or prevent the development of lung granulomas in a mouse model of sarcoidosis

Background

Remodeling of lung tissues during the process of granuloma formation requires significant restructuring of the extra-cellular matrix and cathepsins K, L and S are among the strongest extra-cellular matrix degrading enzymes. Cathepsin K is highly expressed in various pathological granulomatous infiltrates and all three enzymes in their active form are detected in bronchoalveolar lavage fluids from patients with sarcoidosis. Granulomatous inflammation is driven by T-cell response and cathepsins S and L are actively involved in the regulation of antigen presentation and T-cell selection. Here, we show that the disruption of the activities of cathepsins K, L, or S affects the development of lung granulomas in a mouse model of sarcoidosis.

Methods

Apolipoprotein E-deficient mice lacking cathepsin K or L were fed Paigen diet for 16 weeks and lungs were analyzed and compared with their cathepsin-expressing littermates. The role of cathepsin S in the development of granulomas was evaluated using mice treated for 8 weeks with a potent and selective cathepsin S inhibitor.

Results

When compared to wild-type litters, more cathepsin K-deficient mice had lung granulomas, but individually affected mice developed smaller granulomas that were present in lower numbers. The absence of cathepsin K increased the number of multinucleated giant cells and the collagen content in granulomas. Cathepsin L deficiency resulted in decreased size and number of lung granulomas. Apoe-/- mice treated with a selective cathepsin S inhibitor did not develop lung granulomas and only individual epithelioid cells were observed.

Conclusions

Cathepsin K deficiency affected mostly the occurrence and composition of lung granulomas, whereas cathepsin L deficiency significantly reduced their number and cathepsin S inhibition prevented the formation of granulomas.     

Increased Iron Stores Correlate with Worse Disease Outcomes in a Mouse Model of Schistosomiasis Infection

Schistosomiasis is a significant parasitic infection creating disease burden throughout many of the world''s developing nations. Iron deficiency anemia is also a significant health burden resulting from both nutritional deficit as well as parasitic infection in these countries. In this study we investigated the relationships between the disease outcomes of Schistosoma japonicum infection and iron homeostasis. We aimed to determine if host iron status has an effect on schistosome maturation or egg production, and to investigate the response of iron regulatory genes to chronic schistosomiasis infection. Wild-type C57BL/6 and Transferrin Receptor 2 null mice were infected with S. japonicum, and sacrificed at the onset of chronic disease. Transferrin Receptor 2 null mice are a model of type 3 hereditary hemochromatosis and develop significant iron overload providing increased iron stores at the onset of infection. The infectivity of schistosomes and egg production was assessed along with the subsequent development of granulomas and fibrosis. The response of the iron regulatory gene Hepcidin to infection and the changes in iron status were assessed by real-time PCR and Western blotting. Our results show that Hepcidin levels responded to the changing iron status of the animals, but were not significantly influenced by the inflammatory response. We also show that with increased iron availability at the time of infection there was greater development of fibrosis around granulomas. In conclusion, our studies indicate that chronic inflammation may not be the primary cause of the anemia seen in schistosomiasis, and suggest that increased availability of iron, such as through iron supplementation, may actually lead to increased disease severity.