Recognition of PAMPs by immune cells is the first step to start the immune response, whose objective is to eliminate the pathogen and to restore the homeostasis [39, 40]. Innate immunity receptors, such as TLRs, are sensors that detect molecules expressed by pathogens, such as LPS. This molecule is the main component of the outer membrane of Gram-negative bacteria, and is classically recognized by TLR4 and the CD14 co-receptor [10, 41]. The role of CD14 in the outcome of infections by Gram-negative bacteria is controversial and may be related to the induction of a protective immune response or exacerbation of inflammation [23, 28]. In this study, we characterized for the first time the deleterious role of CD14 in pulmonary infection induced by A. xylosoxidans, an emerging nosocomial bacillus whose host responses are not yet to be elucidated.
Previous studies have shown that in pulmonary diseases induced by infection there is an increase in membrane and soluble CD14 expression [42–44]. However, there are conflicting results, reinforcing the fact that CD14 expression and its role varies according to the stimulus and site of infection [23, 28]. Some studies show that CD14 has a protective role in the intestine, by increasing the intestinal barrier function in a colitis model [45], and by controlling the bacterial burden of Shigella flexneri [46]. In contrast, in sepsis models, in LPS-induced endotoxemia or lung infections, CD14 activation appears to have a deleterious role [25, 26, 47, 48], despite a study showing that in cecal ligation and puncture (CLP)-induced sepsis the P2X7 purinergic receptor maintains high levels of CD14 in serum, which contributes to the control of bacterial dissemination [49]. Here, we show that beside A. xylosoxidans inducing a significant increase in the expression of CD14 in the lung parenchyma (at 1 and 3 d.p.i.), its absence attenuates inflammation, as observed by the decreases in leukocyte influx, production of inflammatory mediators, lung damage and lipocalin-2. Lipocalin-2, also known as neutrophil gelatinase-associated lipocalin (NGAL), is a protein normally present in neutrophil granules, but can also be expressed in macrophages, and it is associated with antibacterial activity and intense injury and inflammation [50]. Interestingly, CD14 absence did not interfere with the bacterial load, suggesting that CD14 is more related to the induction of the inflammatory process than to bacterial clearance in this model. Similarly, CD14-deficient mice with pneumonia caused by Acinetobacter baumanni also show a reduction in polymorphonuclear cells (PMNs) and TNF-α in the BALF, but, contrary to our findings, there is an increase in the bacterial load in the lungs in the first hours after infection, indicating that CD14 maybe relevant only in the first hours to control bacterial burden [51]. Interesting, CD14-deficient neutrophils infected with Burkholderia pseudomallei have the same phagocytic capacity as neutrophils from non-deficient animals, suggesting that the CD14-dependent bacterial load reduction mechanism does not occur via phagocytosis and killing capacity [48], but it is associated with increased release of trappin-2 in the lungs, an essential antimicrobial peptide in the elimination of P. aeruginosa [52].
The influx of neutrophils into the lungs is important for the control of an infection, but if exacerbated, it can contribute to lung damage through the release of NETs and enzymes with proteolytic activity, such as neutrophil elastase [53], or the release of lipocalin-2 and other molecules that increase chemokine production by PMNs [54]. After A. xylosoxidans infection, we observed that CD14-deficient mice show a reduction in lipocalin-2, as well as a lower number of neutrophils in the lungs, in addition to less pulmonary damage, resulting in longer survival rate than wild-type animals. In agreement with our results, the lung parenchyma of CD14-deficient animals is more preserved during S. pneumoniae infection when compared to WT mice, in addition to having a lower concentration of total proteins in the lungs, and a reduction in IL-1β, IL-6 and CXCL-1 in lungs and plasma of infected mice [55]. In tuberculosis, CD14 also mediates PMN recruitment and release of pro-inflammatory cytokines in the lungs, culminating in lung injury and higher animal mortality [56].
CD14 was first described as anchored to the membrane of macrophages and monocytes, but after inflammatory stimuli such as LPS, CD14 can be released in soluble form via the action of proteases or even after escaping from binding to GPI in post-translational modifications [33, 57]. Defining a pattern of CD14 expression in leukocytes from pneumonia patients or from infected mice is a challenge. Patients with tuberculosis have lower CD14 expression on monocytes when compared to healthy individuals [58], and similarly, mCD14 expression is reduced in alveolar macrophages from mice infected with E. coli when compared to cells from wild-type mice [59]. In contrast, the expression of mCD14 on monocytes and macrophages of children with pneumonia is higher when compared to the control group [60]. In our model of pulmonary infection by A. xylosoxidans, although we did not observe an increase in CD14 MFI on the surface of the evaluated leukocytes, we found an increase in the absolute number of neutrophils and macrophages expressing CD14 that are recruited to the lung, suggesting that the increase in cells capable of signaling via mCD14 contribute to the exacerbation of pulmonary inflammation. sCD14 is considered a potential marker of pneumonia in children [60] and a predictor of severity of coronavirus disease 2019 (COVID-19) [61]. Furthermore, sCD14 is considered an acute phase marker [38], and it was even shown that this molecule is capable of inducing pro-inflammatory cytokines independent of LPS in CF patients, contributing to the persistence of inflammation in these patients [62, 63]. Corroborating these findings, we observed that A. xylosoxidans induces sCD14 release in the BALF and serum of infected animals, suggesting that part of mCD14 of leukocytes or others cells, such epithelial cells, is shedding to the soluble profile during infection. The dynamics of sCD14 release by other leukocytes and non-immune cells needs to be further investigated.
Taken together, our findings highlight the detrimental role of CD14 in A. xylosoxidans-induced pneumonia, mediated by pulmonary and systemic release of sCD14, which likely maintains leukocyte recruitment to the lungs and impairs pulmonary homeostasis. Furthermore, these results reinforce that CD14 inhibition may be a possible alternative therapy for patients with chronic lung inflammation caused by A. xylosoxidans.