Th1, Th17, and Th1Th17 Lymphocytes during Tuberculosis: Th1 Lymphocytes Predominate and Appear as Low-Differentiated CXCR3+CCR6+ Cells in the Blood and Highly Differentiated CXCR3+/-CCR6- Cells in the Lungs.

Th1 lymphocytes are considered the main mediators of protection against tuberculosis (TB); however, their phenotypic characteristics and relationship with Th17 and Th1Th17 populations during TB are poorly understood. We have analyzed Th1, Th17, and Th1Th17 lymphocytes in the blood and pulmonary lesions of TB patients. The populations were identified based on the production of IFN-γ and/or IL-17 and the coexpression of CXCR3 (X3) and CCR6 (R6). In the blood, IL-17+ and IFN-γ+IL-17+ lymphocytes were barely detectable (median, <0.01% of CD4+ lymphocytes), whereas IFN-γ+ lymphocytes predominated (median, 0.45%). Most IFN-γ+ lymphocytes (52%) were X3+R6+, suggesting their "nonclassical" (ex-Th17) nature. In the lungs, IL-17+ and IFN-γ+IL-17+ lymphocytes were more frequent (0.3%, p < 0.005), yet IFN-γ+ cells predominated (11%). Phenotypically, lung CD4+ cells were X3+/loR6- The degree of differentiation of blood effector CD4+ lymphocytes (evaluated based on CD62L/CD27/CD28 coexpression) increased as follows: X3+R6+ < X3+R6- < X3-R6-, with X3-R6- cells being largely terminally differentiated CD62L-CD27-CD28- cells. Lung CD4+ lymphocytes were highly differentiated, recalling blood X3+/-R6- populations. Following in vitro stimulation with anti-CD3/anti-CD28 Abs, X3+R6+CD4+ lymphocytes converted into X3+R6- and X3-R6- cells. The results demonstrate that, during active TB, Th1 lymphocytes predominate in blood and lungs, document differences in X3/R6 expression by blood and lung CD4+ cells, and link the pattern of X3/R6 expression with the degree of cell differentiation. These findings add to the understanding of immune mechanisms operating during TB and are relevant for the development of better strategies to control it.

Gr-1dimCD11b+ immature myeloid-derived suppressor cells but not neutrophils are markers of lethal tuberculosis infection in mice.

Tuberculosis (TB) disease may progress at different rates and have different outcomes. Neutrophils have been implicated in TB progression; however, data on their role during TB are controversial. In this study, we show that in mice, TB progression is associated with the accumulation of cells that express neutrophilic markers Gr-1 and Ly-6G but do not belong to conventional neutrophils. The cells exhibit unsegmented nuclei, have Gr-1(dim)Ly-6G(dim)CD11b(+) phenotype, and express F4/80, CD49d, Ly-6C, CD117, and CD135 markers characteristic not of neutrophils but of immature myeloid cells. The cells accumulate in the lungs, bone marrow, spleen, and blood at the advanced (prelethal) stage of Mycobacterium tuberculosis infection and represent a heterogeneous population of myeloid cells at different stages of their differentiation. The accumulation of Gr-1(dim)CD11b(+) cells is accompanied by the disappearance of conventional neutrophils (Gr-1(hi)Ly-6G(hi)-expressing cells). The Gr-1(dim)CD11b(+) cells suppress T cell proliferation and IFN-γ production in vitro via NO-dependent mechanisms, that is, they exhibit characteristics of myeloid-derived suppressor cells. These results document the generation of myeloid-derived suppressor cells during TB, suggesting their role in TB pathogenesis, and arguing that neutrophils do not contribute to TB pathology at the advanced disease stage.

In mice, tuberculosis progression is associated with intensive inflammatory response and the accumulation of Gr-1 cells in the lungs.

BACKGROUND: Infection with Mycobacterium tuberculosis (Mtb) results in different clinical outcomes ranging from asymptomatic containment to rapidly progressing tuberculosis (TB). The mechanisms controlling TB progression in immunologically-competent hosts remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: To address these mechanisms, we analyzed TB progression in a panel of genetically heterogeneous (A/SnxI/St) F2 mice, originating from TB-highly-susceptible I/St and more resistant A/Sn mice. In F2 mice the rates of TB progression differed. In mice that did not reach terminal stage of infection, TB progression did not correlate with lung Mtb loads. Nor was TB progression correlated with lung expression of factors involved in antibacterial immunity, such as iNOS, IFN-gamma, or IL-12p40. The major characteristics of progressing TB was high lung expression of the inflammation-related factors IL-1beta, IL-6, IL-11 (p<0.0003); CCL3, CCL4, CXCL2 (p<0.002); MMP-8 (p<0.0001). The major predictors of TB progression were high expressions of IL-1beta and IL-11. TNF-alpha had both protective and harmful effects. Factors associated with TB progression were expressed mainly by macrophages (F4-80(+) cells) and granulocytes (Gr-1(hi)/Ly-6G(hi) cells). Macrophages and granulocytes from I/St and A/Sn parental strains exhibited intrinsic differences in the expression of inflammatory factors, suggesting that genetically determined peculiarities of phagocytes transcriptional response could account for the peculiarities of gene expression in the infected lungs. Another characteristic feature of progressing TB was the accumulation in the infected lungs of Gr-1(dim) cells that could contribute to TB progression. CONCLUSIONS/SIGNIFICANCE: In a population of immunocompetent hosts, the outcome of TB depends on quantitatively- and genetically-controlled differences in the intensity of inflammatory responses, rather than being a direct consequence of mycobacterial colonization. Local accumulation of Gr-1(dim) cells is a newly identified feature of progressing TB. High expression of IL-1beta and IL-11 are potential risk factors for TB progression and possible targets for TB immunomodulation.

A human-like TB in genetically susceptible mice followed by the true dormancy in a Cornell-like model.

Mouse tuberculosis (TB) models that utilize genetically susceptible mouse strains demonstrate many features of human lung disease. In the present study, pathology caused by progressive M. tuberculosis H37Rv infection in TB-susceptible I/St mice following the low-dose aerosol challenge showed close similarity to human TB, with formation of necrotic granuloma with adjusting B-cell-rich follicles. A remarkable feature was the development of hypoxic zones around TB lesions by day 60 of infection. Necrotizing inflammatory foci were abundantly infiltrated with Ly-6G+ neutrophils. The levels of mRNA for neutrophil-recruiting factors (KC, MIP-2, IL-17 and IL-6) were all significantly increased in infected compared to naïve animals. A profound elevation of the mRNA level for IFN-gamma resulted neither in mycobacterial growth inhibition, nor in IL-17 response counter-regulation. Three-month therapy with RIF and INH resulted in eradication of culturable mycobacteria (at least 9 months following withdrawal), recovery of the lung tissue structure, and normalization of inflammatory genes expression. However, stable mycobacterial DNA (M. tuberculosis-specific insertion IS6110 detected by the qrt-PCR) was retained in the lungs for a long time after culturable bacilli were eliminated, and combination of lung homogenate liquid cultures with auramine staining demonstrated the presence of acid-fast bacilli with unaltered mycobacterial morphology. The lack of mycobacterial growth on agar, their microscopic detection in concentrated liquid cultures, and the increase in numbers of IS6110 copies in vivo at late stages of cured infection suggest that in our model dormant M. tuberculosis survived in the host.

Multigenic control of disease severity after virulent Mycobacterium tuberculosis infection in mice.

Following challenge with virulent Mycobacterium tuberculosis, mice of the I/St inbred strain exhibit shorter survival time, more rapid body weight loss, higher mycobacterial loads in organs, and more severe lung histopathology than mice of the A/Sn strain. We previously performed a genome-wide scan for quantitative trait loci (QTLs) that control the severity of M. tuberculosis-triggered disease in [(A/Sn x I/St) F1 x I/St] backcross-1 (BC1) mice and described several QTLs that are significantly or suggestively linked to body weight loss. In the present study we expanded our analysis by including the survival time phenotype and by genotyping 406 (A/Sn x I/St) F2 mice for the previously identified chromosomal regions of interest. The previously identified 12-cM-wide QTL on distal mouse chromosome 3 was designated tbs1 (tuberculosis severity 1); the location of the QTL on proximal chromosome 9 was narrowed to a 9-cM interval, and this QTL was designated tbs2. Allelic variants of the tbs2 locus appeared to be involved in control of both body weight loss and survival time. Also, the data strongly suggested that a QTL located in the vicinity of the H-2 complex on chromosome 17 is involved in control of tuberculosis in mice of both genders, whereas the tbs1 locus seemed to have an effect on postinfection body weight loss in female mice. Interestingly, these loci appeared to interact with each other, which suggests that there might be a basic genetic network for the control of intracellular parasites. Overall, linkage data reported here for F2 mice are in agreement with, and add to, our previous findings concerning the control of M. tuberculosis-triggered disease in the BC1 segregation.