Tuberculous pleurisy allows the study of specific cells at the site of Mycobacterium tuberculosis infection. Among pleural lymphocytes, natural killer (NK) cells are a major source of interferon γ ...(IFN-γ), and their functions are regulated by activating and inhibitory receptors. Programmed death-1 (PD-1), programmed death ligand 1 (PD-L1), and programmed death ligand 2 (PD-L2) are recognized inhibitory receptors in adaptive immunity, but their role during innate immunity remains poorly understood. We investigated the PD-1:PDL1/PD-L2 pathway on NK cell effector functions in peripheral blood and pleural fluid from patients with tuberculosis. M. tuberculosis stimulation significantly up-regulated PD-1, PD-L1, and PD-L2 levels on NK cells. Interestingly, a direct correlation between PD-1 and IFN-γ expression on NK cells was observed. Moreover, blockade of the PD-1 pathway markedly augmented lytic degranulation and IFN-γ production of NK cells against M. tuberculosis. Furthermore, PD-1+ NK cells displayed a diminished IFN-γ mean fluorescence intensity, denoting the relevance of PD-1 on IFN-γ regulation. Together, we described a novel inhibitory role played by PD-1:PD-L interactions in innate immunity in tuberculosis.
Protective immunity against Mycobacterium tuberculosis requires the generation of cell-mediated immunity. We investigated the expression and role of programmed death 1 (PD-1) and its ligands, ...molecules known to modulate T cell activation, in the regulation of IFN-gamma production and lytic degranulation during human tuberculosis. We demonstrated that specific Ag-stimulation increased CD3+PD-1+ lymphocytes in peripheral blood and pleural fluid from tuberculosis patients in direct correlation with IFN-gamma production from these individuals. Moreover, M. tuberculosis-induced IFN-gamma participated in the up-regulation of PD-1 expression. Blockage of PD-1 or PD-1 and its ligands (PD-Ls: PD-L1, PD-L2) enhanced the specific degranulation of CD8+ T cells and the percentage of specific IFN-gamma-producing lymphocytes against the pathogen, demonstrating that the PD-1:PD-Ls pathway inhibits T cell effector functions during active M. tuberculosis infection. Furthermore, the simultaneous blockage of the inhibitory receptor PD-1 together with the activation of the costimulatory protein signaling lymphocytic activation molecule led to the promotion of protective IFN-gamma responses to M. tuberculosis, even in patients with weak cell-mediated immunity against the bacteria. Together, we demonstrated that PD-1 interferes with T cell effector functions against M. tuberculosis, suggesting that PD-1 has a key regulatory role during the immune response of the host to the pathogen.
Mycobacterium tuberculosis expands CD4+IFN‐γ+IL‐17+ lymphocytes in direct correlation with the severity of active disease.
Th1 lymphocytes are crucial in the immune response against Mycobacterium ...tuberculosis. Nevertheless, IFN‐γ alone is not sufficient in the complete eradication of the bacteria, suggesting that other cytokines might be required for pathogen removal. Th17 cells have been associated with M. tuberculosis infection, but the role of IL‐17‐producing cells in human TB remains to be understood. Therefore, we investigated the induction and regulation of IFN‐γ and IL‐17 during the active disease. TB patients were classified as High and Low Responder individuals according to their T cell responses against the antigen, and cytokine expression upon M. tuberculosis stimulation was investigated in peripheral blood and pleural fluid. Afterwards, the potential correlation among the proportions of cytokine‐producing cells and clinical parameters was analyzed. In TB patients, M. tuberculosis induced IFN‐γ and IL‐17, but in comparison with BCG‐vaccinated healthy donors, IFN‐γ results were reduced significantly, and IL‐17 was markedly augmented. Moreover, the main source of IL‐17 was represented by CD4+IFN‐γ+IL‐17+ lymphocytes, a Th1/Th17 subset regulated by IFN‐γ. Interestingly, the ratio of antigen‐expanded CD4+IFN‐γ+IL‐17+ lymphocytes, in peripheral blood and pleural fluid from TB patients, was correlated directly with clinical parameters associated with disease severity. Indeed, the highest proportion of CD4+IFN‐γ+IL‐17+ cells was detected in Low Responder TB patients, individuals displaying severe pulmonary lesions, and longest length of disease evolution. Taken together, the present findings suggest that analysis of the expansion of CD4+IFN‐γ+IL‐17+ T lymphocytes in peripheral blood of TB patients might be used as an indicator of the clinical outcome in active TB.
Two metallo-β-lactamase-producing
Klebsiella pneumoniae
(HA30 and HA31) were isolated in a hospital in Argentina during 2018.
K. pneumoniae
HA30 was isolated from a rectal swab during the ...epidemiological surveillance for carbapenemase-producing strains, while
K. pneumoniae
HA31 was collected from the same patient 4 days after hospitalization. The aim of the present study was to identify the clonal relationships and resistome of these two NDM-producing
K. pneumoniae
strains isolated from a patient with a fatal outcome. Whole-genome sequencing (WGS) was performed using Illumina MiSeq-I, and subsequent analysis involved genome assembly, annotation, antibiotic resistance gene identification, multilocus sequence typing (MLST), and plasmid characterization using bioinformatics tools. Conjugation assays to
E. coli
J53 was conducted as previously described.
K. pneumoniae
HA30 exhibited extensively drug-resistant phenotype, while HA31 was multidrug-resistant as defined by Magiorakos et al., including both resistance to carbapenems, aminoglycosides and ciprofloxacin with
bla
NDM-5
,
bla
CTX-M-15
and
rmtB
genes found in both strains. MLST analysis showed that both strains belonged to ST11, differing by only 4 cgSNPs, indicating that
K. pneumoniae
HA30 and HA31 were the same strain. Conjugation assays revealed that
K. pneumoniae
HA31 strain possessed a transferable plasmid to
E. coli
J53. Bioinformatics studies identified that the same strain colonizing an inpatient during hospital admission subsequently caused the infection leading to a fatal outcome, being the first report of
bla
NDM-5
,
rmtB
and
bla
CTX-M-15
genes in a
K. pneumoniae
ST11 strain from Latin America. Our results also highlighted the importance of focusing on epidemiological surveillance programs.
Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated ...the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen.
We evaluated the role of regulatory T cells (CD4⁺ CD25⁺ Foxp3⁺ cells, Tregs) in human Mycobacterium tuberculosis infection. Tregs were expanded in response to M. tuberculosis in healthy tuberculin ...reactors, but not in tuberculin-negative individuals. The M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) resulted in regulatory T cell expansion, whereas the M. tuberculosis 19-kDa protein and heat shock protein 65 had no effect. Anti-IL-10 and anti-TGF-β alone or in combination, did not reduce expansion of Tregs. In contrast, the cyclooxygenase enzyme-2 inhibitor NS398 significantly inhibited expansion of Tregs, indicating that prostaglandin E2 (PGE2) contributes to Treg expansion. Monocytes produced PGE2 upon culturing with heat-killed M. tuberculosis or ManLAM, and T cells from healthy tuberculin reactors enhanced PGE2 production by monocytes. Expanded Tregs produced significant amounts of TGF-β and IL-10 and depletion of Tregs from PBMC of these individuals increased the frequency of M. tuberculosis-responsive CD4⁺ IFN-γ cells. Culturing M. tuberculosis-expanded Tregs with autologous CD8⁺ cells decreased the frequency of IFN-γ⁺cells. Freshly isolated PBMC from tuberculosis patients had increased percentages of Tregs, compared to healthy tuberculin reactors. These findings demonstrate that Tregs expand in response to M. tuberculosis through mechanisms that depend on ManLAM and PGE2.
Alterations of myeloid cell populations have been reported in patients with tuberculosis (TB). In this work, we studied the relationship between myeloid-derived suppressor cells (MDSC) and monocytes ...subsets with the immunological responsiveness of TB patients. Individuals with active TB were classified as low responders (LR-TB) or high responders (HR-TB) according to their T cell responses against a cell lysate of
(
-Ag). Thus, LR-TB, individuals with severe disease, display a weaker immune response to
compare to HR-TB, subjects with strong immunity against the bacteria. We observed that LR-TB presented higher percentages of CD16 positive monocytes as compared to HR-TB and healthy donors. Moreover, monocyte-like (M-MDSC) and polymorphonuclear-like (PMN-MDSC) MDSC were increased in patients and the proportion of M-MDSC inversely correlated with IFN-γ levels released after
-Ag stimulation in HR-TB. We also found that LR-TB displayed the highest percentages of circulating M-MDSC. These results demonstrate that CD16 positive monocytes and M-MDSC frequencies could be used as another immunological classification parameter. Interestingly, in LR-TB, frequencies of CD16 positive monocytes and M-MDSC were restored after only three weeks of anti-TB treatment. Together, our findings show a link between the immunological status of TB patients and the levels of different circulating myeloid cell populations.
According to the World Health Organization, carbapenem-resistant
Enterobacteriaceae
(CRE) belong to the highest priority group for the development of new antibiotics. Argentina-WHONET data showed ...that Gram-negative resistance frequencies to imipenem have been increasing since 2010 mostly in two CRE bacteria:
Klebsiella pneumoniae
and
Enterobacter cloacae
Complex (ECC). This scenario is mirrored in our hospital. It is known that
K. pneumoniae
and the ECC coexist in the human body, but little is known about the outcome of these species producing KPC, and colonizing or infecting a patient. We aimed to contribute to the understanding of the rise of the ECC in Argentina, taking as a biological model both a patient colonized with two KPC-producing strains (one
Enterobacter hormaechei
and one
K. pneumoniae
) and
in vitro
competition assays with prevalent KPC-producing ECC (KPC-ECC) versus KPC-producing
K. pneumoniae
(KPC-Kp) high-risk clones from our institution. A KPC-producing
E. hormaechei
and later a KPC-Kp strain that colonized a patient shared an identical novel conjugative IncM1 plasmid harboring
bla
KPC-2
. In addition, a total of 19 KPC-ECC and 58 KPC-Kp strains isolated from nosocomial infections revealed that high-risk clones KPC-ECC ST66 and ST78 as well as KPC-Kp ST11 and ST258 were prevalent and selected for competition assays. The competition assays with KCP-ECC ST45, ST66, and ST78 versus KPC-Kp ST11, ST18, and ST258 strains analyzed here showed no statistically significant difference. These assays evidenced that high-risk clones of KPC-ECC and KPC-Kp can coexist in the same hospital environment including the same patient, which explains from an ecological point of view that both species can exchange and share plasmids. These findings offer hints to explain the worldwide rise of KPC-ECC strains based on the ability of some pandemic clones to compete and occupy a certain niche. Taken together, the presence of the same new plasmid and the fitness results that showed that both strains can coexist within the same patient suggest that horizontal genetic transfer of
bla
KPC-2
within the patient cannot be ruled out. These findings highlight the constant interaction that these two species can keep in the hospital environment, which, in turn, can be related to the spread of KPC.
Production of IFN‐γ contributes to host defense against Mycobacterium tuberculosis (Mtb) infection. We previously demonstrated that Signaling lymphocytic activation molecule‐associated protein (SAP) ...expression on cells from tuberculosis (TB) patients was inversely correlated with IFN‐γ production. Here we first investigated the role of NK, T‐ and B‐cell antigen (NTB‐A)/SAP pathway in the regulation of Th1 response against Mtb. Upon antigen stimulation, NTB‐A phosphorylation rapidly increases and afterwards modulates IFN‐γ and IL‐17 secretion. To sustain a healthy immune system, controlled expansion and contraction of lymphocytes, both during and after an adaptive immune response, is essential. Besides, restimulation‐induced cell death (RICD) results in an essential homeostatic mechanism for precluding excess T‐cell accumulation and associated immunopathology during the course of certain infections. Accordingly, we found that the NTB‐A/SAP pathway was required for RICD during active tuberculosis. In low responder (LR) TB patients, impaired RICD was associated with diminished FASL levels, IL‐2 production and CD25high expression after cell‐restimulation. Interestingly, we next observed that SAP mediated the recruitment of the Src‐related kinase FYNT, only in T cells from LR TB patients that were resistant to RICD. Together, we showed that the NTB‐A/SAP pathway regulates T‐cell activation and RICD during human TB. Moreover, the NTB‐A/SAP/FYNT axis promotes polarization to an unfavorable Th2‐phenotype.
:Enterobacter cloacae complex (ECC) has lately awakened interest due to its increasing resistance to carbapenems codified by several genes all over the globe. Even though there are some sequence ...types (ST) which represent high-risk clones, there is substantial clonal diversity in the ECC. This work aimed to perform whole-genome sequencing (WGS), genomic analysis and phylogenetic studies of a KPC-producing multidrug-resistant (MDR) ECC isolate from Argentina.
: We analysed the genome of an MDR KPC-producing ECC strain isolated from a urine sample from a patient in a hospital in Argentina. The WGS was done by Illumina MiSeq-I. The genome was assembled with SPAdes 3.9.0, and annotated with PROKKA, RAST, and Blast. Plasmids were identified with PlasmidFinder. Antibiotic resistance genes were detected using RESfinder, CARD, and Blastn. STs were identified with pubMLST.
: The strain was identified as Enterobacter hormaechei which is an important emerging human pathogen. No ST could be assigned; 6 out of 7 alleles of multilocus sequence typing (MLST) were the same as for E. hormaechei ST66, which is a high-risk clone. We found multiple acquired antibiotic resistance genes including blaKPC-2 in an IncM1 plasmid, and a secretion system VI, which can favour the prevalence of ECC strains while competing with other bacteria.
: Due to its MLST profile being so close to E. hormaechei ST66, the acquisition of multiple resistance genes, and the presence of the secretion systems, the potential of this strain for becoming a new high-risk clone cannot be discarded.
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