Currently, protective and inflammatory responses during CBPP are not fully understood. In recent years, IFN-γ and especially IFN-γ-secreting CD4+
T cells have been proposed as key elements required for protection [10
], as their levels were inversely correlated with severity of disease. A recent study employing murine CD4+
specific mAb to deplete bovine CD4+
T cells, including the IFN-γ-secreting population, represents the first attempt to investigate, in vivo
, the role of CD4+
T cells in a primary CBPP infection [5
]. Animals were depleted at 6 dpi, since the time window of complete depletion is less than two weeks and we wanted to time depletion in the onset of clinical disease. It cannot be ruled out that CD4+
T cells contributed to CBPP-specific cytokine responses before depletion. Four out of twenty animals had to be euthanized before the envisaged end of the trial because of ethical reasons. This is in the range of reported mortality of CBPP especially given the fact that those animals were kept at ideal conditions with food ad libitum
and no climate or production-associated stress. The animals were monitored for selected in vivo
cytokine plasma profiles during Mycoplasma mycoides
infection to see whether the depletion of most CD4+
specific T cells over a time window had any effects on the cytokine profiles. Flow cytometry analysis had demonstrated that the CD4+
T cell depletion was successful and that naïve CD4+
T cells reappeared approximately ten days post depletion, but did not reach the levels of control animals until the end of the experiment [5
Acute and sub-acute cases of CBPP are characterised by strong inflammatory reaction leading to serofibrinous pleuropneumonia, pleural effusion and tissue damage. We expected a correlation between increased TNF-α plasma levels and severe clinical signs and/or lesions, since TNF-α is one of the key inflammatory cytokines that it is released in large amounts following infection with gram-negative and other bacteria including mycoplasmas and is responsible for systemic complications [11
]. An increase of TNF-α in the plasma was observed in most of the animals following infection, which supports the hypothesis that TNF-α mediates inflammation in CBPP. The levels did not increase dramatically, which might be attributed to the fact that TNF-α is produced locally, has a short half-life, and so primarily accomplishes its effects locally. Therefore, the increase of the plasma levels might be a signature of what is happening in certain regions of the lung.
The high degree of lung lobulation and the absence of collateral airways in the bovine lung often quarantine inflammatory processes to defined lobes [12
] and prevent an inflammation of the entire lung as seen only in a small fraction of the animals after experimental infection. The most severely infected animals showed a significantly higher level of plasma TNF-α when compared to the animals showing a milder reaction. However, it must be noted that only three out of four animals most severely affected showed very high concentrations of the cytokine. Conversely, one animal with a mild form of disease had a very high plasma concentration of TNF-α throughout the experiment. The data further suggest that the CD4+
T cell depletion did not have a significant effect on the TNF-α plasma levels. This is not surprising, as cells from the macrophage lineage, which are amply represented by alveolar macrophages, are important producers of TNF-α.
Although IFN-γ is known to prime macrophages to secrete more TNF-α after stimulation, the IFN-γ in this experiment frequently peaked later (BD 91, BD 93, BD 95, BD 98, BD 107, BD 118) rather than simultaneously with TNF-α. This time difference suggests that early TNF-α secretion is not highly dependent on plasma IFN-γ. Again, local IFN-γ release in the lungs, which could not be detected given the set-up of this experiment, might have different kinetics and affected TNF-α concentrations. It has been shown in vitro
that direct exposure of bovine alveolar macrophages to Mycoplasma mycoides
can trigger TNF-α secretion [8
]. In vivo
IFN-γ measurement showed increased release of the cytokine in plasma from the depleted group 16 dpi (ten days post CD4+
T cell depletion). During this period the percentage of CD4+
cells was less than 1% as demonstrated by flow cytometric analysis. The CD4+
T cells that re-appeared derived most likely from the thymus and were naïve, thus not capable of mounting a prompt immune response [13
]. This implies that CD4+
T cells are not the main or most important source of IFN-γ in the course of CBPP, and that other cellular compartments must be responsible for IFN-γ production. Natural killer cells, CD8+
T cells or γδ T cells are also capable of producing IFN-γ, but their role in CBPP immune response has not been elucidated.
Animals that had to be euthanized because of severe clinical signs had significantly higher levels of plasma IFN-γ in comparison to animals with low pathology scores. On the other hand, animals with low pathology scores did not show high IFN-γ responses.
This was surprising; since previous studies had shown that higher IFN-γ levels in stimulated cultures of peripheral blood mononuclear cells (PBMCs) correlated with milder disease [10
]. Other studies based on similar in vitro
cell culture assays did not confirm the positive correlation between resistance and IFN-γ recall responses from PBMCs [14
]. While TNF-α and IFN-γ may have a protective effect or stimulate protective responses, their concentrations may be linked to the severity of the infection and the amount of mycoplasma in the host. Bovine IL-4 is not a Th2 signature cytokine but is associated to a Th2 profile [15
Interestingly, measurable IL-4 plasma concentrations were only detected in one non-CD 4+
T cell-depleted animal (BD97) that developed severe CBPP infection. This animal also presented high IFN-γ levels suggesting that acute and severe CBPP cases develop an imbalanced cytokine release with high level of IFN-γ and IL-4 resulting in severe pathology as reported in other cattle diseases and in some cases of human mycobacterial infection [16
Analysis of the anti-inflammatory cytokine IL-10, which increased in most of the animals between 6 and 13 dpi, showed an interesting correlation between augmented release of IL-10 and increased plasma levels of TNF-α or IFN-γ. Since most of the animals showing increasing IL-10 concentrations had pathomorphological signatures of CBPP, we speculate that the increase in IL-10 is not sufficient to prevent excessive inflammatory processes that shape CBPP lung lesions. Future experiments should include the monitoring of this cytokine in immune animals. Our findings did not indicate a prominent role for CD4+
T cells in IL-10 secretion and it is known that many populations of bovine cells, including Th0, Th1 and Th2 cells, can produce IL-10 [17
]. Stimulated human alveolar macrophages can be induced to produce IL-10 [18
], but similar data for cattle are missing.
We characterized IgG1, IgG2, IgA, and IgM antibody responses against Mycoplasma antigen post infection. As we did not evaluate the binding affinities of the secondary reagents we were not able to draw meaningful conclusions regarding a T1 or T2 bias based upon the ratio of IgG1 to IgG2 titres [15
]. We observed statistically higher IgG1 and IgG2 responses (see Additional file 6
: Observed qualitative Mycoplasma-specific antibody responses) in severely affected animals. Future studies should evaluate the biological role of these responses, in particular a possible involvement of immune complexes in disease progression. Immune complexes are likely to be formed during CBPP as a result of high levels of immune globulins in combination with proinflammatory cytokines. This is the first study looking for cytokine signatures in cattle that have been infected with Mycoplasma mycoides
. Future studies should focus on whether the cytokine kinetics observed are the cause of pathoimmunological reactions or a manifestation of it in order to understand the disease, design proper control measures and direct the immune responses of next generation vaccines properly.