Infection with hypervirulent Mycobacterium tuberculosis triggers emergency myelopoiesis but not trained immunity.

Introduction: During infection, bone marrow (BM) hematopoiesis is reprogrammed toward myeloid cell production, a mechanism named emergency myelopoiesis. In addition to replenishing myeloid cells, emergency myelopoiesis has been linked to trained immunity, a process that allows enhanced innate immune responses to secondary challenges. Although hematopoietic alterations during tuberculosis (TB) have been described and Mycobacterium tuberculosis may colonize the BM, studies using the mouse model of infection and the laboratory reference strain M. tuberculosis H37Rv have demonstrated limited emergency myelopoiesis and trained immunity. Methods: To further address this issue, we aerosol- infected C57BL/6 mice with high doses of the hypervirulent M. tuberculosis isolate HN878 and monitored alterations to the BM. This experimental model better resembles the human blood immune signature of TB. Results and discussion: We found increased frequencies of lineage-Sca-1+cKit+ (LSK) cells and the granulocyte/macrophage progenitor (GMP) population. At the mature cell level, we observed an increase of monocytes and neutrophils in the blood and lung, likely reflecting the increased BM myeloid output. Monocytes or monocyte-derived macrophages recovered from the BM of M. tuberculosis HN878-infected mice did not show signs of trained immunity, suggesting an uncoupling of emergency myelopoiesis and trained immunity in the BM. Surprisingly, M. tuberculosis HN878-induced emergency myelopoiesis was not fully dependent on IFNγ, as mice lacking this cytokine and infected under the same conditions as wild-type mice still presented BM alterations. These data expand our understanding of the immune response to M. tuberculosis and raise awareness of pathogen strain-imposed differences to host responses.

Physical Interactions With Bacteria and Protozoan Parasites Establish the Scavenger Receptor SSC4D as a Broad-Spectrum Pattern Recognition Receptor.

Since the pioneering discoveries, by the Nobel laureates Jules Hoffmann and Bruce Beutler, that Toll and Toll-like receptors can sense pathogenic microorganisms and initiate, in vertebrates and invertebrates, innate immune responses against microbial infections, many other families of pattern recognition receptors (PRRs) have been described. One of such receptor clusters is composed by, if not all, at least several members of the scavenger receptor cysteine-rich (SRCR) superfamily. Many SRCR proteins are plasma membrane receptors of immune cells; however, a small subset consists of secreted receptors that are therefore in circulation. We here describe the first characterization of biological and functional roles of the circulating human protein SSC4D, one of the least scrutinized members of the family. Within leukocyte populations, SSC4D was found to be expressed by monocytes/macrophages, neutrophils, and B cells, but its production was particularly evident in epithelial cells of several organs and tissues, namely, in the kidney, thyroid, lung, placenta, intestinal tract, and liver. Similar to other SRCR proteins, SSC4D shows the capacity of physically binding to different species of bacteria, and this opsonization can increase the phagocytic capacity of monocytes. Importantly, we have uncovered the capacity of SSC4D of binding to several protozoan parasites, a singular feature seldom described for PRRs in general and here demonstrated for the first time for an SRCR family member. Overall, our study is pioneer in assigning a PRR role to SSC4D.

Myeloid HIF-1α regulates pulmonary inflammation during experimental Mycobacterium tuberculosis infection.

The transcription factor hypoxia-inducible factor-1 alpha (HIF-1α) is a key regulator of the response and function of myeloid cells in hypoxic and inflammatory microenvironments. To define the role of HIF-1α in tuberculosis, the progression of aerosol Mycobacterium tuberculosis infection was analysed in mice deficient in HIF-1α in the myeloid lineage (mHIF-1α-/- ). We show that myeloid HIF-1α is not required for the containment of the infection, as both wild-type (WT) and mHIF-1α-/- mice mounted normal Th1 responses and maintained control of bacterial growth throughout infection. However, during chronic infection mHIF-1α-/- mice developed extensive lymphocytic inflammatory involvement of the interstitial lung tissue and died earlier than WT mice. These data support the hypothesis that HIF-1α activity coordinates the response of myeloid cells during M. tuberculosis infection to prevent excessive leucocyte recruitment and immunopathological consequences to the host.

TNF-Mediated Compensatory Immunity to Mycobacterium avium in the Absence of Macrophage Activation by IFN-γ.

Granuloma formation is a hallmark of several infectious diseases, including those caused by Mycobacterium sp These structures are composed of accumulations of inflammatory cells, and it has been shown that cytokines such as IFN-γ and TNF-α are required for granuloma assembly during M. avium infections in mice. Macrophages (MΦs) insensitive to IFN-γ (MIIG) mice have MΦs, monocytes, and dendritic cells that are unresponsive to IFN-γ. We observed that although IFN-γ-/- mice present an exacerbated infection, the same is not true for MIIG animals, where the same levels of protection as the wild-type animals were observed in the liver and partial protection in the spleen. Unlike IFN-γ-/- mice, MIIG mice still develop well-defined granulomas, suggesting that IFN-γ-mediated MΦ activation is not required for granuloma assembly. This work also shows that MIIG animals exhibit increased cell recruitment with higher CD4+ T cells numbers as well as increased IFN-γ and TNF-α expression, suggesting that TNF-α may have a role in protection and may compensate the lack of MΦ response to IFN-γ in the MIIG model. TNF-α-deficient MIIG mice (MIIG.TNF-α-/-) exhibited increased bacterial burdens when compared with MIIG mice. These results suggest that in the absence of IFN-γ signaling in MΦs, TNF-α has a protective role against M. avium.

Targeted macrophages delivery of rifampicin-loaded lipid nanoparticles to improve tuberculosis treatment.

AIM: This work aims to develop a mannosylated nanostructured lipid carrier (NLC) loaded with rifampicin to improve tuberculosis treatment. MATERIALS & METHODS: An active targeting strategy was used and the nanoparticles were characterized. Effects on cell viability and the antimycobacterial activity of the nanoformulations were evaluated. RESULTS: The nanoparticles developed exhibited a size of about 315 nm and polydispersity <0.2. The drug encapsulation efficiency was higher than 90% and its release was sensitive to pH. The mannosylated NLCs showed efficient uptake by bone marrow derived macrophages. Further, rifampicin-loaded mannosylated NLCs were more efficient in inducing a decrease of intracellular growth of mycobacteria. CONCLUSION: The NLCs developed can be used as a promising carrier for safer and efficient management of tuberculosis.

Innate IFN-γ-Producing Cells Developing in the Absence of IL-2 Receptor Common γ-Chain.

IFN-γ is known to be predominantly produced by lymphoid cells such as certain subsets of T cells, NK cells, and other group 1 innate lymphoid cells. In this study, we used IFN-γ reporter mouse models to search for additional cells capable of secreting this cytokine. We identified a novel and rare population of nonconventional IFN-γ-producing cells of hematopoietic origin that were characterized by the expression of Thy1.2 and the lack of lymphoid, myeloid, and NK lineage markers. The expression of IFN-γ by this population was higher in the liver and lower in the spleen. Furthermore, these cells were present in mice lacking both the Rag2 and the common γ-chain (γc) genes (Rag2-/-γc-/-), indicating their innate nature and their γc cytokine independence. Rag2-/-γc-/- mice are as resistant to Mycobacterium avium as Rag2-/- mice, whereas Rag2-/- mice lacking IFN-γ are more susceptible than either Rag2-/- or Rag2-/-γc-/- These lineage-negative CD45+/Thy1.2+ cells are found within the mycobacterially induced granulomatous structure in the livers of infected Rag2-/-γc-/- animals and are adjacent to macrophages that expressed inducible NO synthase, suggesting a potential protective role for these IFN-γ-producing cells. Accordingly, Thy1.2-specific mAb administration to infected Rag2-/-γc-/- animals increased M. avium growth in the liver. Overall, our results demonstrate that a population of Thy1.2+ non-NK innate-like cells present in the liver expresses IFN-γ and can confer protection against M. avium infection in immunocompromised mice.

Lack of the Transcription Factor Hypoxia-Inducible Factor 1α (HIF-1α) in Macrophages Accelerates the Necrosis of Mycobacterium avium-Induced Granulomas.

The establishment of mycobacterial infection is characterized by the formation of granulomas, which are well-organized aggregates of immune cells, namely, infected macrophages. The granuloma's main function is to constrain and prevent dissemination of the mycobacteria while focusing the immune response to a limited area. In some cases these lesions can grow progressively into large granulomas which can undergo central necrosis, thereby leading to their caseation. Macrophages are the most abundant cells present in the granuloma and are known to adapt under hypoxic conditions in order to avoid cell death. Our laboratory has developed a granuloma necrosis model that mimics the human pathology of Mycobacterium tuberculosis, using C57BL/6 mice infected intravenously with a low dose of a highly virulent strain of Mycobacterium avium. In this work, a mouse strain deleted of the hypoxia inducible factor 1α (HIF-1α) under the Cre-lox system regulated by the lysozyme M gene promoter was used to determine the relevance of HIF-1α in the caseation of granulomas. The genetic ablation of HIF-1α in the myeloid lineage causes the earlier emergence of granuloma necrosis and clearly induces an impairment of the resistance against M. avium infection coincident with the emergence of necrosis. The data provide evidence that granulomas become hypoxic before undergoing necrosis through the analysis of vascularization and quantification of HIF-1α in a necrotizing mouse model. Our results show that interfering with macrophage adaptation to hypoxia, such as through HIF-1α inactivation, accelerates granuloma necrosis.