Browsing by Subject "Innate immune system"

Now showing 1 - 2 of 2

Open Access
Characterization of altered innate and adaptive immune responses of primary immune deficient patients
(2021-05) İbibik, Bilgehan
The immune system dysregulations led to primary immune deficiencies (PIDs). Immune deficiencies can be divided into two groups, primary and secondary immune deficiencies. The reasons for primary immune deficiencies could be inherited immune dysfunctions originated from autosomal recessive or autosomal dominant mutations. Combinations of ongoing infections, lymphoproliferation, atopies, malignancies, autoimmunity and granulomatous processes are seen in primary immune deficiency disorders. To establish distinctive therapies, characterization of immune deficiencies along with the understanding the course of impaired mechanisms are very critical to offer robust means of cure. In this study, the effects of different mutations (RLTPR, RLTPR-TLR1, and CTLA-4) on innate and adaptive immune systems were investigated. RLTPR (CARMIL2) is a cytosolic scaffold protein which facilitates CD28 co-stimulation for T-cell activation. RLTPR facilitates recruitment of CARMA1, a cytosolic adaptor, along with the BCL10 and MALT1 which form CBM complex to CD28 site to activate NF-κB signaling pathway. Besides facilitating CD28 co-stimulation, RLTPR takes place in cell shape control, phagocytosis and endocytosis movements by promoting actin polymerization. Reduced CD4+ T-cells, memory B-cells and antibody response along with the impaired B-cell receptor mediated NF-κB activation were mainly observed in RLTPR deficient patients. Moreover, polarity and migration of the T-cells were affected by RLTPR deficiency in patients. Herein, immunological phenotypes of RLTPR deficient patient with and without CMV infection were described. As expected, Treg counts of the patient were reduced as compared to healthy controls both with and without infection. Although Treg cell counts of patient were decreased, IL-10 secretion upon cytosolic nucleic acid ligands was increased in infection due to ongoing defense and homeostasis. Under CMV infection, patient showed elevated IL-1β and TNF-α responses upon activation of TLRs and cytosolic nucleic acid sensors. However, without any viral infections, TLR7 and TLR8 mediated IL-1β and TNF-α responses were impaired in patient. Moreover, under CMV infection, TLR and cytosolic nucleic acid sensors mediated antiviral IFN-α, IFN-γ and IL-12 responses were substantially increased compared to healthy subjects. Unexpectedly, when patient PBMCs were assessed following infection, we detect that IFN-α and IFN-γ levels of the patient in response to endosomal TLRs and cytosolic nucleic acids stimulations were reduced. Throughout the course of viral infection due to ongoing defense by innate immune cells, one would predict to detect higher type I and II IFN responses. When infection was cleared with medical treatment, it is observed that not only endosomal TLRs but also the cytosolic nucleic acid sensors were impaired in RLTPR patient which makes the patient vulnerable to viral infections. Thereafter, altered immune responses of RLTPR-TLR1 deficient patient was investigated. Decreased number of Treg cells from whole blood of the patient was confirmed. IL-1β response from PBMCs of patient was elevated by stimulation with the TLR and cytosolic nucleic acid ligands. Especially, TLR2-6 response was elevated which could be the result of TLR1 deficiency because immune response could be compensated when TLR1 is deficient but TLR6 is not. Also, remaining improved proinflammatory cytokine response to different PRR ligands could be reasoned by ongoing infection. IFN-α secretion was increased by endosomal TLR and cytosolic nucleic acid ligands while IL-12 secretion of patient showed ligand specific modulated responses which suggest an ongoing infection. As expected, there was no detectable TLR1 mediated IL-12 response due to TLR1 deficiency. However, TLR2-6 mediated IL-12 secretion was elevated in patient compared to healthy subjects which could be regarded as a compensatory response against TLR1 deficiency. RLTPR-TLR1 deficient patient PBMCs elicited elevated IL-10 response to TLR and cytosolic nucleic acid ligand triggering even though Treg cells were reduced., implying that other suppressor cells could be involved in this response. CTLA-4, a negative regulator of T-cells, is expressed on activated T-cells and Treg cells. CTLA-4 binds to CD80/86 molecules on APCs which prevents CD28 co-stimulation. Without CD28 co-stimulation, the T-cell cannot be activated. Patients with insufficient CTLA-4 receptors could have an increased number of Treg cells with reduced function. CTLA-4 haploinsufficiency leads to hyperactive T-cells, enhanced autoreactive B-cells and reduced numbers of circulating B-cells. In this work, immune responses of four CTLA-4 patients were also studied. Patient #1 showed higher CD4+/CD8+ ratio, elevated LDG frequency, as well as reduced TCR expression on CD3+ cells and elevated pAKT protein levels. Patient #2 had increased LDG count, reduced pDC and Treg population in whole blood. Similar blood cell profile to Patient #2, Patient #3 additionally had increased monocyte percentage and lower CD4+/CD8+ ratio which could indicate an ongoing infection. Patient #4 showed decreased pDC, Treg counts, increased levels of PD-L1 on CD8+ cells, Treg cells and B-cells, reduced TCR expression on CD3+ T-cells, increased pAKT and p4EBP1 levels which all may contribute to compensate the autoimmune status of the CTLA-4 mutation. B-cell percentages and CTLA-4 expression levels of all patients were not altered while mTOR, pmTOR, STAT3, pSTAT3, AKT, p4EBP1 and HIf-1α expression levels were impaired in all patients. Collectively, our findings imply that the complexity of the dysregulation of these deficiencies, and point-out to an unappreciated immune functional status of these patients. We propose that investigation of the innate immune arm of these individuals which were perceived as solely related to and impacting only adaptive immune system is necessary to offer more robust therapies to these patients.
Open Access
Characterization of innate and adaptive immune responses of two rare primary immune deficiencies: CTPS1 and CD55
(2019-09) Kaya, Göksu Gökberk
Primary Immune deficiencies (PIDs) are disorders of immune system caused by mutated genes. There are approximately 350 different disorders and each day novel ones are being defined. They can be categorized based on part of immune system harboring mutation; that is, they can be divided into disorders of innate and adaptive immune system. Each of them represents itself distinctly. In that perspective, studies based on characterization of PIDs enable us to comprehend how immune system works. Herein, we characterized innate and adaptive immune responses of two rare immune deficiencies: CTPS1 and CD55 which are novel examples of disorders of adaptive and innate immune system, respectively. CTPS1 is an enzyme functioning in de novo synthesis of nucleotide, CTP. Defective CTPS1 enzyme impairs lymphocytes to proliferate, however, other aspects of this deficiency still remain elusive. Since patients are prone to viral infections, we first explored functionality of cytotoxic T-cells through assessing STAT1 phosphorylation levels and expression of activation markers. Even though flow cytometry analyses revealed that CTPS1 deficient CD8+ T-cells had normal phospho-STAT1 levels, degranulation marker confined to surface of CD8+ Tcells were found to be elevated. Next, we investigated CD4+ T-cells with cytokines that are crucial for differentiation and fate. We detected that patient CD4+ T-cells had low phospho-STAT3 and phospho-STAT5 levels. Then, we checked the cytokine production profiles of CD4+ T-cells. Data indicated that percentages of IL-17a and IL 10 secreting cells are reduced in patient whereas Th1 and Th2 signatures were similar to healthy controls. Moreover, IFN-a levels of PBMCs upon TLR3, TLR7 and TLR9 ligand stimulations were found to be similar to healthy responses. Notably, patient had slightly reduced TLR7 and IFI16-STING mediated type II IFN secretion. We further showed that CTPS1 PBMCs had normal IL-12 levels, implying that the reduction in IFN-g was not due to either dysfunction of innate immune cells or by aberrant APC function. Surprisingly, patient PBMCs had higher number of granulocytes and flow cytometry analyses revealed that these granulocytes were CD14- CD15+ low-density granulocytes. This prompted us to assess the NETotic tendencies of CTPS1 neutrophils and we observed via microscopic and spectrofluorometric investigations that they underwent spontaneous NETosis. In the second part of this study, we worked with CD55 deficient patient PBMCs. CD55 is a complement regulatory protein and it inhibits formation of C3-convertase in classical and alternative complement pathways. Thus, patients suffer from aberrant complement activation in its absence as well as severe bowel inflammation and recurrent infections along with nutrient loss leading to malnutrition and growth deprivation. Eculizumab therapy was initiated to these patients in order to neutralize their pathologic C5 levels. We attempted to investigate effect of CD55 deficiency on PRR-complement cross-talk, recurrent infections and checked the contribution of Eculizumab therapy to their immune status. PBMCs of 4 patients, i) before (BT) and ii) after (AT) a single dose of Eculizumab administration were isolated. BT PBMCs had significantly reduced IFN-a and IP-10 secretions upon endosomal (TLR3, TLR7 and TLR9) TLRs and nucleic acid sensors (STING, DAI, RIG-I & MAVS) stimulations. Moreover, single Eculizumab therapy did not alter this innate immune dysfunction. Furthermore, we assessed levels of TNF-a, IL-6 productions from PBMCs stimulated with same ligands and observed that IL-6 but not TNF-a was reduced after PRR stimulations. Next, immunomodulatory effects of CD55 EVs before and after Eculizumab therapy was sought. ELISA results demonstrated that AT EV incubation on CD55-/- PBMCs lead to reduced TNF-a, IL-1b and IFN-g production. Meanwhile, AT EVs increased IL-10 production from patient PBMCs. Lastly, we assessed cytokine levels after healthy PBMCs were incubated with BT and AT EVs and found that PBMCs that incubated with BT EVs had elevated levels of IP-10 cytokine. When taken together, progression of PIDs might have been contributed by extracellular vesicles.